Method for administering a cytokine to the central nervous system and the lymphatic system

ABSTRACT

The present invention is directed to a method for delivering cytokines to the central nervous system and the lymphatic system by way of a tissue innervated by the trigeminal nerve and/or olfactory nerve. Cytokines include tumor necrosis factors, interleukins, interferons, particularly interferon-β and its muteins such as IFN-β ser17 . Such a method of delivery can be useful in the treatment of central nervous system disorders, brain disorders, proliferative, viral, and/or autoimmune disorders such as Sjogren&#39;s disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/733,168, filed Dec. 8, 2000, which is hereby incorporated herein inits entirety by reference.

FIELD OF THE INVENTION

The present invention is directed to a method for delivering cytokinesto the central nervous system and by the lymphatic system by way of atissue innervated by the trigeminal nerve and/or olfactory nerve.Cytokines include tumor necrosis factors, interleukins, interferons,particularly β-interferon and its muteins such as IFN-β_(ser17). Such amethod of delivery can be useful in the treatment of central nervoussystem and/or brain disorders.

BACKGROUND OF THE INVENTION

The central nervous system (CNS) includes several tissues and organs,such as the brain, the brain stem, and the spinal cord. Each of theseorgans and tissues is made up of a variety of different types of cellsand subcellular structures, e.g., neurons, glial cells, dendrites,axons, myelin, and various membranes. The CNS is isolated from theexternal world by several membranes that both cushion and protect theseorgans, tissues, cells, and structures. For example, the membranes thatform the blood-brain barrier protect the brain from certain contents ofthe blood. The blood-cerebrospinal fluid barrier protects other portionsof the CNS from many chemicals and microbes.

Access to the CNS for some substances is provided by specialized activetransport systems or through passive diffusion through the protectivemembrane into the CNS. Present methods for delivering desiredtherapeutic agents to the CNS are typically invasive. For example, apump implanted into the chest cavity (an intracerebroventricular pump)can effectively deliver a variety of useful compounds to the brain.However, implanting such a pump requires surgery, which can entail avariety of serious complications. Certain compounds (e.g., epiduralpainkillers) can be injected directly through the protective membraneinto the CNS. Such injection is, however, impractical for mostmedications. Better methods for administering desired agents to the CNS,brain, spinal cord, and lymphatic channels are needed.

SUMMARY OF THE INVENTION

The present invention relates to a method for transporting or deliveringa cytokine, such as an interferon, an interleukin, or a tumor necrosisfactor, preferably interferon-β, to the central nervous system of asubject. The method employs administration of the cytokine to a tissueinnervated by the trigeminal nerve and/or olfactory nerve.

In one embodiment, the method administers the cytokine through themucosa or epithelium of the nasal cavity, tongue, mouth, skin, orconjunctiva. In another embodiment, the method includes administering acomposition of the cytokine to the nasal cavity, under the tongue, tothe skin, or to the conjunctiva of the subject. The cytokine can then beabsorbed through a mucosa or epithelium and transported to the centralnervous system of the mammal.

In another embodiment, the method includes administering the cytokine ina manner such that the cytokine is absorbed through the tissue andtransported into the central nervous system of the mammal by a neuralpathway and in an amount effective to provide a protective ortherapeutic effect on a cell of the central nervous system.

The present invention further relates to a method for transporting ordelivering a cytokine, such as an interferon, an interleukin, or a tumornecrosis factor, preferably interferon-β, to the lymphatic system of asubject. The method employs administration of the cytokine to a tissueinnervated by the trigeminal nerve and/or olfactory nerve.

In another embodiment, the method includes administering the cytokine ina manner such that the cytokine is absorbed through the tissue andtransported into the central nervous system of the mammal by a neuralpathway and in an amount effective to modulate an immune or inflammatoryresponse.

In other embodiments, the method of administering a cytokine is used forthe treatment and/or prevention of central nervous system disorders,brain disorders, proliferative, viral, and/or autoimmune disorders.

The composition can be of any form suitable for administration by theseroutes and can include a carrier that facilitates absorption of thecytokine, transport of the cytokine by a neural pathway, and/ortransport of the cytokine to the lymphatic system, CNS, brain, and/orspinal cord. Preferred compositions include one or more of a solubilityenhancing additive, a hydrophilic additive, an absorption promotingadditive, a cationic surfactant, a viscosity enhancing additive, or asustained release matrix or composition, a lipid-based carrier,preferably a micellar or liposomal composition, a bilayer destabilizingadditive, or a fusogenic additive. The composition can be formulated asa cosmetic for dermal delivery.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the level of Betaseron in the blood stream over timefollowing both intravenous administration (I.V.) and intranasaladministration (I.N.) in a rat.

DETAILED DESCRIPTION OF THE INVENTION

Routes of Administration

The method of the invention administers the cytokine to tissueinnervated by the trigeminal and olfactory nerves. Such nerve systemscan provide a direct connection between the outside environment and thebrain, thus providing advantageous delivery of a cytokine to the CNS,including brain, brain stem, and/or spinal cord. Cytokines are unable tocross or inefficiently cross the blood-brain barrier from thebloodstream into the brain. The methods of the present invention allowfor the delivery of the cytokine by way of the olfactory and/ortrigeminal nerve rather than through the circulatory system. This methodof administration allows for the efficient delivery of a cytokine to theCNS, brain, or spinal cord.

The Olfactory Nerve

The method of the invention includes administration of a cytokine totissue innervated by the olfactory nerve. Preferably, the cytokine isdelivered to the olfactory area in the upper third of the nasal cavityand particularly to the olfactory epithelium.

Fibers of the olfactory nerve are unmyelinated axons of olfactoryreceptor cells that are located in the superior one-third of the nasalmucosa. The olfactory receptor cells are bipolar neurons with swellingscovered by hair-like cilia that project into the nasal cavity. At theother end, axons from these cells collect into aggregates and enter thecranial cavity at the roof of the nose. Surrounded by a thin tube ofpia, the olfactory nerves cross the subarachnoid space containing CSFand enter the inferior aspects of the olfactory bulbs. Once the cytokineis dispensed into the nasal cavity, the cytokine can undergo transportthrough the nasal mucosa and into the olfactory bulb and interconnectedareas of the brain, such as the hippocampal formation, amygdaloidnuclei, nucleus basalis of Meynert, locus ceruleus, the brain stem, andthe like.

The Trigeminal Nerve

The method of the invention administers the cytokine to tissueinnervated by the trigeminal nerve. The trigeminal nerve innervatestissues of a mammal's (e.g., human) head including skin of the face andscalp, oral tissues, and tissues of and surrounding the eye. Thetrigeminal nerve has three major branches, the ophthalmic nerve, themaxillary nerve, and the mandibular nerve. The method of the inventioncan administer the cytokine to tissue innervated by one or more of thesebranches.

The Ophthalmic Nerve and its Branches

The method of the invention can administer the cytokine to tissueinnervated by the ophthalmic nerve branch of the trigeminal nerve. Theophthalmic nerve innervates tissues including superficial and deep partsof the superior region of the face, such as the eye, the lacrimal gland,the conjunctiva, and skin of the scalp, forehead, upper eyelid, andnose.

The ophthalmic nerve has three branches known as the nasociliary nerve,the frontal nerve, and the lacrimal nerve. The method of the inventioncan administer the cytokine to tissue innervated by the one or more ofthe branches of the ophthalmic nerve. The frontal nerve and its branchesinnervate tissues including the upper eyelid, the scalp, particularlythe front of the scalp, and the forehead, particularly the middle partof the forehead. The nasociliary nerve forms several branches includingthe long ciliary nerves, the ganglionic branches, the ethmoidal nerves,and the infratrochlear nerve. The long ciliary nerves innervate tissuesincluding the eye. The posterior and anterior ethmoidal nerves innervatetissues including the ethmoidal sinus and the inferior two-thirds of thenasal cavity. The infratrochlear nerve innervates tissues including theupper eyelid and the lacrimal sack. The lacrimal nerve innervatestissues including the lacrimal gland, the conjunctiva, and the uppereyelid. Preferably, the present method administers the cytokine to theethmoidal nerve.

The Maxillary Nerve and its Branches

The method of the invention can administer the cytokine to tissueinnervated by the maxillary nerve branch of the trigeminal nerve. Themaxillary nerve innervates tissues including the roots of several teethand facial skin, such as skin on the nose, the upper lip, the lowereyelid, over the cheekbone, over the temporal region. The maxillarynerve has branches including the infraorbital nerve, thezygomaticofacial nerve, the zygomaticotemporal nerve, the nasopalatinenerve, the greater palatine nerve, the posterior superior alveolarnerves, the middle superior alveolar nerve, and the interior superioralveolar nerve. The method of the invention can administer the cytokineto tissue innervated by the one or more of the branches of the maxillarynerve.

The infraorbital nerve innervates tissue including skin on the lateralaspect of the nose, upper lip, and lower eyelid. The zygomaticofacialnerve innervates tissues including skin of the face over the zygomaticbone (cheekbone). The zygomaticotemporal nerve innervates tissueincluding the skin over the temporal region. The posterior superioralveolar nerves innervate tissues including the maxillary sinus and theroots of the maxillary molar teeth. The middle superior alveolar nerveinnervates tissues including the mucosa of the maxillary sinus, theroots of the maxillary premolar teeth, and the mesiobuccal root of thefirst molar tooth. The anterior superior alveolar nerve innervatestissues including the maxillary sinus, the nasal septum, and the rootsof the maxillary central and lateral incisors and canine teeth. Thenasopalantine nerve innervates tissues including the nasal septum. Thegreater palatine nerve innervates tissues including the lateral wall ofthe nasal cavity. Preferably, the present method administers thecytokine to the nasopalatine nerve and/or greater palatine nerve.

The Mandibular Nerve and its Branches

The method of the invention can administer the cytokine to tissueinnervated by the mandibular nerve branch of the trigeminal nerve. Themandibular nerve innervates tissues including the teeth, the gums, thefloor of the oral cavity, the tongue, the cheek, the chin, the lowerlip, tissues in and around the ear, the muscles of mastication, and skinincluding the temporal region, the lateral part of the scalp, and mostof the lower part of the face.

The mandibular nerve has branches including the buccal nerve, theauriculotemporal nerve, the inferior alveolar nerve, and the lingualnerve. The method of the invention can administer the cytokine to one ormore of the branches of the mandibular nerve. The buccal nerveinnervates tissues including the cheek, particularly the skin of thecheek over the buccinator muscle and the mucous membrane lining thecheek, and the mandibular buccal gingiva (gum), in particular theposterior part of the buccal surface of the gingiva. Theauriculotemporal nerve innervates tissues including the auricle, theexternal acoustic meatus, the tympanic membrane (eardrum), and skin inthe temporal region, particularly the skin of the temple and the lateralpart of the scalp. The inferior alveolar nerve innervates tissuesincluding the mandibular teeth, in particular the incisor teeth, thegingiva adjacent the incisor teeth, the mucosa of the lower lip, theskin of the chin, the skin of the lower lip, and the labial mandibulargingivae. The lingual nerve innervates tissues including the tongue,particularly the anterior two-thirds of the tongue, the floor of themouth, and the gingivae of the mandibular teeth. Preferably, the methodof the invention administers the cytokine to one or more of the inferioralveolar nerve, the buccal nerve, and/or the lingual nerve.

Tissues Innervated by the Trigeminal Nerve

The method of the invention can administer the cytokine to any of avariety of tissues innervated by the trigeminal nerve. For example, themethod can administer the cytokine to skin, epithelium, or mucosa of oraround the face, the eye, the oral cavity, the nasal cavity, the sinuscavities, or the ear.

Preferably, the method of the invention administers the cytokine to skininnervated by the trigeminal nerve. For example, the present method canadminister the cytokine to skin of the face, scalp, or temporal region.Suitable skin of the face includes skin of the chin; the upper lip, thelower lip; the forehead, particularly the middle part of the forehead;the nose, including the tip of the nose, the dorsum of the nose, and thelateral aspect of the nose; the cheek, particularly the skin of thecheek over the buccinator muscle or skin over the cheek bone; skinaround the eye, particularly the upper eyelid and the lower eyelid; or acombination thereof. Suitable skin of the scalp includes the front ofthe scalp, scalp over the temporal region, the lateral part of thescalp, or a combination thereof. Suitable skin of the temporal regionincludes the temple and scalp over the temporal region.

Preferably, the method of the invention administers the cytokine tomucosa or epithelium innervated by the trigeminal nerve. For example,the present method can administer the cytokine to mucosa or epitheliumof or surrounding the eye, such as mucosa or epithelium of the uppereyelid, the lower eyelid, the conjunctiva, the lacrimal system, or acombination thereof. The method of the invention can also administer thecytokine to mucosa or epithelium of the sinus cavities and/or nasalcavity, such as the inferior two-thirds of the nasal cavity and thenasal septum. The method of the invention can also administer thecytokine to mucosa or epithelium of the oral cavity, such as mucosa orepithelium of the tongue; particularly the anterior two-thirds of thetongue and under the tongue; the cheek; the lower lip; the upper lip;the floor of the oral cavity; the gingivae (gums), in particular thegingiva adjacent the incisor teeth, the labial mandibular gingivae, andthe gingivae of the mandibular teeth; or a combination thereof.Preferably, the method of the invention administers the cytokine tomucosa or epithelium of the nasal cavity. Other preferred regions ofmucosa or epithelium for administering the cytokine include the tongue,particularly sublingual mucosa or epithelium, the conjunctiva, thelacrimal system, particularly the palpebral portion of the lacrimalgland and the nasolacrimal ducts, the mucosa of the lower eyelid, themucosa of the cheek, or a combination thereof.

Preferably, the method of the invention administers the cytokine tonasal tissues innervated by the trigeminal nerve. For example, thepresent method can administer the cytokine to nasal tissues includingthe sinuses, the inferior two-thirds of the nasal cavity and the nasalseptum. Preferably, the nasal tissue for administering the cytokineincludes the inferior two-thirds of the nasal cavity and the nasalseptum.

Preferably, the method of the invention administers the cytokine to oraltissues innervated by the trigeminal nerve. For example, the presentmethod can also administer the cytokine to oral tissues such as theteeth, the gums, the floor of the oral cavity, the cheeks, the lips, thetongue, particularly the anterior two-thirds of the tongue, or acombination thereof. Suitable teeth include mandibular teeth, such asthe incisor teeth. Suitable portions of the teeth include the roots ofseveral teeth, such as the roots of the maxillary molar teeth, themaxillary premolar teeth, the maxillary central and lateral incisors,the canine teeth, and the mesiobuccal root of the first molar tooth, ora combination thereof. Suitable portions of the lips include the skinand mucosa of the upper and lower lips. Suitable gums include thegingiva adjacent the incisor teeth and the gingivae of the mandibularteeth, such as the labial mandibular gingivae, or a combination thereof.Suitable portions of the cheek include the skin of the cheek over thebuccinator muscle, the mucous membrane lining the cheek, and themandibular buccal gingiva (gum), in particular the posterior part of thebuccal surface of the gingiva, or a combination thereof. Preferred oraltissue for administering the cytokine includes the tongue, particularlysublingual mucosa or epithelium, the mucosa inside the lower lip, themucosa of the cheek, or a combination thereof.

Preferably, the method of the invention administers the cytokine to atissue of or around the eye that is innervated by the trigeminal nerve.For example, the present method can administer the cytokine to tissueincluding the eye, the conjunctiva, and the lacrimal gland including thelacrimal sack, the skin or mucosa of the upper or lower eyelid, or acombination thereof. Preferred tissue of or around the eye foradministering the cytokine includes the conjunctiva, the lachrimalsystem, the skin or mucosa of the eyelid, or a combination thereof.Cytokine that is administered conjunctivally but not absorbed throughthe conjunctival mucosa can drain through nasolachrimal ducts into thenose, where it can be transported to the CNS, brain, and/or spinal cordas though it had been intranasally administered.

Preferably, the method of the invention administers the cytokine to atissue of or around the ear that is innervated by the trigeminal nerve.For example, the present method can administer the cytokine to tissueincluding the auricle, the external acoustic meatus, the tympanicmembrane (eardrum), and the skin in the temporal region, particularlythe skin of the temple and the lateral part of the scalp, or acombination thereof. Preferred tissue of or around the ear foradministering the cytokine includes the skin of the temple.

Cytokines

Cytokines can be administered to the CNS, brain, and/or spinal cordaccording to the present invention. Cytokines that can be administeredby the method of the invention are cytokines that are immunomodulators,such as interleukins (i.e., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9 and IL-10), interferons, and tumor necrosis factor (i.e.,TNF-α and TNF-β), and that have activities directed at cells of theimmune system. These cytokines are of interest as therapeutic cytokines,for example, for treatment of viral diseases and control of cancer. Itis believed that such cytokines have not been observed to haveneurotrophic activity, or to have other direct, beneficial effects onneurons characteristic of nerve growth factor and like compounds. Thus,it was not expected that such cytokines should be transported into theCNS, brain, and or spinal cord, particularly not by a neural pathway, orfrom tissues innervated by the olfactory and/or trigeminal nerves.

A preferred cytokine for use in the practice of the invention aremembers of the interferon family. Interferons (IFNs) are a family ofmolecules encompassing over 20 different proteins and are members of thecytokine family that induce antiviral, antiproliferative, antitumor,and/or cytokine effects. IFNs are relatively small species-specific,single chain polypeptides, which are produced in response to a varietyof inducers, such as mitogens, polypeptides, viruses, and the like. Inhumans, IFNs are produced in forms α, β, γ, ω, and τ. Syntheticinterferons are also known in the art. See, for example, U.S. Pat. No.6,114,145, herein incorporated by reference. Upon secretion frommammalian cells, interferon molecules bind to a receptor on the surfaceof a target cell and elicit a chain of events, which can alter theamount and activity of protein in the target cell. Such alterations caninclude, for example, changes in gene transcription or enzymaticactivity. A preferred interferon for use in the practice of theinvention is interferon-β (IFN-β), interferon-α (IFN-α), andinterferon-γ (IFN-γ).

Biologically active variants of cytokines are also encompassed by themethod of the present invention. Such variants should retain thebiological activity of the cytokine. For example, when the cytokine isan interferon, such as IFN-α, IFN-β, IFN-γ, the ability to bind theirrespective receptor sites will be retained. Such activity may bemeasured using standard bioassays. Representative assays detecting theability of the variant to interact with an interferon receptor type Ican be found in, for example, U.S. Pat. No. 5,766,864, hereinincororpated by reference. Preferably, the variant has at least the sameactivity as the native molecule. Alternatively, the biological activityof a variant of the cytokine of the invention can be assayed bymeasuring the ability of the variant to increase viral resistance in acell line using a standard viral reduction assay. See for example, U.S.Pat. No. 5,770,191, herein incorporated by reference. Other assays forbiological activity include, anti-proliferative assays as described inU.S. Pat. No. 5,690,925.

Suitable biologically active variants can be fragments, analogues, andderivatives of the cytokine polypeptides. By “fragment” is intended aprotein consisting of only a part of the intact cytokine polypeptidesequence. The fragment can be a C-terminal deletion or N-terminaldeletion of the cytokine polypeptide. By “analogue” is intended ananalogue of either the full length polypeptide having biologicalactivity or a fragment thereof, that includes a native sequence andstructure having one or more amino acid substitutions, insertions, ordeletions. Peptides having one or more peptoids (peptide mimics) arealso encompassed by the term analogue (see i.e., InternationalPublication No. WO 91/04282). By “derivative” is intended any suitablemodification of the native polypeptide or fragments thereof, or theirrespective analogues, such as glycosylation, phosphorylation, or otheraddition of foreign moieties, so long as the activity is retained.

Preferably, naturally or non-naturally occurring variants of a cytokinehave amino acid sequences that are at least 70%, preferably 80%, morepreferably, 85%, 90%, 91%, 92%, 93%, 94% or 95% identical to the aminoacid sequence to the reference molecule, for example, the native humaninterferon, or to a shorter portion of the reference interferonmolecule. More preferably, the molecules are 96%, 97%, 98% or 99%identical. Percent sequence identity is determined using theSmith-Waterman homology search algorithm using an affine gap search witha gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrixof 62. The Smith-Waterman homology search algorithm is taught in Smithand Waterman, Adv. Appl. Math. (1981) 2:482-489. A variant may, forexample, differ by as few as 1 to 10 amino acid residues, such as 6-10,as few as 5, as few as 4, 3, 2, or even 1 amino aid residue.

With respect to optimal alignment of two amino acid sequences, thecontiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference amino acid sequence will include atleast 20 contiguous amino acid residues, and may be 30, 40, 50, or moreamino acid residues. Corrections for sequence identity associated withconservative residue substitutions or gaps can be made (seeSmith-Waterman homology search algorithm).

The art provides substantial guidance regarding the preparation and useof such variants, as discussed further below. A fragment of a cytokinepolypeptide will generally include at least about 10 contiguous aminoacid residues of the full-length molecule, preferably about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably about 20-50 or more contiguous amino acid residues offull-length cytokine polypeptide.

For example, conservative amino acid substitutions may be made at one ormore predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a cytokine, such as an interferon (i.e.,IFN-α, IFN-β, or IFN-γ) without altering its biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif.

Alternatively, variant cytokine nucleotide sequences can be made byintroducing mutations randomly along all or part of a cytokine codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for cytokine biological activity to identify mutantsthat retain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques described herein.

Alternatively, the cytokine can be synthesized chemically, by any ofseveral techniques that are known to those skilled in the peptide art.See, for example, Li et al. (1983) Proc. Natl. Acad. Sci. USA80:2216-2220, Steward and Young (1984) Solid Phase Peptide Synthesis(Pierce Chemical Company, Rockford, Ill.), and Baraney and Merrifield(1980) The Peptides: Analysis, Synthesis, Biology, ed. Gross andMeinhofer, Vol. 2 (Academic Press, New York, 1980), pp. 3-254,discussing solid-phase peptide synthesis techniques; and Bodansky (1984)Principles of Peptide Synthesis (Springer-Verlag, Berlin) and Gross andMeinhofer, eds. (1980) The Peptides: Analysis, Synthesis, Biology, Vol.1 (Academic Press, New York), discussing classical solution synthesis.The cytokine can also be chemically prepared by the method ofsimultaneous multiple peptide synthesis. See, for example, Houghten(1984) Proc. Natl. Acad. Sci. USA 82:5131-5135; and U.S. Pat. No.4,631,211.

The cytokine used in the methods of the invention can be from any animalspecies including, but not limited to, avian, canine, bovine, porcine,equine, and human. Preferably, the cytokine is from a mammalian specieswhen the cytokine is to be used in treatment of a mammalian viral,immunomodulatory, or neurologic disorder of the CNS, brain or spinalcord, and more preferably is from a mammal of the same species as themammal undergoing treatment for such a disorder.

Interferon-β

The term “IFN-β” as used herein refers to IFN-β or variants thereof,sometimes referred to as IFN-β-like polypeptides. Human IFN-β variants,which may be naturally occurring (e.g., allelic variants that occur atthe IFN-β locus) or recombinantly produced, have amino acid sequencesthat are the same as, similar to, or substantially similar to the maturenative IFN-β sequence. DNA sequences encoding human IFN-β are alsoavailable in the art. See, for example, Goeddel et al. (1980) NucleicAcid Res. 8:4057 and Tanigachi et al. (1979) Proc. Japan Acad. Sci.855:464. Fragments of IFN-β or truncated forms of IFN-β that retaintheir activity are also encompassed. These biologically active fragmentsor truncated forms of IFN-β are generated by removing amino acidresidues from the full-length IFN-β amino acid sequence usingrecombinant DNA techniques well known in the art. IFN-β polypeptides maybe glycosylated or unglycosylated, as it has been reported in theliterature that both the glycosylated and unglycosylated forms of IFN-βshow qualitatively similar specific activities and that, therefore, theglycosyl moieties are not involved in and do not contribute to thebiological activity of IFN-β.

The IFN-β variants encompassed herein include muteins of the nativemature IFN-β sequence, wherein one or more cysteine residues that arenot essential to biological activity have been deliberately deleted orreplaced with other amino acids to eliminate sites for eitherintermolecular crosslinking or incorrect intramolecular disulfide bondformation. IFN-β variants of this type include those containing aglycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine,histidine, tryptophan, serine, threonine, or methionine substituted forthe cysteine found at amino acid 17 of the mature native amino acidsequence. Serine and threonine are the more preferred replacementsbecause of their chemical analogy to cysteine. Serine substitutions aremost preferred. For example, an IFN-β variant can comprise a serineresidue replacing the cysteine found at amino acid 17 of the maturenative sequence. Cysteine 17 may also be deleted using methods known inthe art (see, for example, U.S. Pat. No. 4,588,585, herein incorporatedby reference), resulting in a mature IFN-β mutein that is one amino acidshorter than the native mature IFN-β. Thus, IFN-β variants with one ormore mutations that improve, for example, their pharmaceutical utilityare also encompassed by the present invention.

The skilled artisan will appreciate that additional changes can beintroduced by mutation into the nucleotide sequences encoding IFN-β,thereby leading to changes in the IFN-β amino acid sequence, withoutaltering the biological activity of the interferon. Thus, an isolatednucleic acid molecule encoding an IFN-β variant having a sequence thatdiffers from human IFN-β can be created by introducing one or morenucleotide substitutions, additions, or deletions into the correspondingnucleotide sequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedIFN-β. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such IFN-βvariants are also encompassed by the present invention. Variants ofIFN-β are described in European Patent Application No. 18545981, andU.S. Pat. Nos. 4,518,584, 4,588,585, and 4,737,462, all of which areincorporated herein by reference.

Biologically active IFN-β variants encompassed by the invention alsoinclude IFN-β polypeptides that have covalently linked with, forexample, polyethylene glycol (PEG) or albumin.

Biologically active variants of IFN-β encompassed by the inventionshould retain IFN-β activities, particularly the ability to bind toIFN-β receptors or retain immunomodulatory or anti-viral activities. Insome embodiments, the IFN-β variant retains at least about 25%, about50%, about 75%, about 85%, about 90%, about 95%, about 98%, about 99% ormore of the biological activity of the native IFN-β polypeptide. IFN-βvariants whose activity is increased in comparison with the activity ofthe native IFN-β polypeptide are also encompassed. The biologicalactivity of IFN-β variants can be measured by any method known in theart. Examples of such assays can be found in Fellous et al. (1982) Proc.Natl. Acad. Sci USA 79:3082-3086; Czerniecki et al. (1984) J. Virol.49(2):490-496; Mark et al. (1984) Proc. Natl. Acad. Sci. USA81:5662-5666; Branca et al. (1981) Nature 277:221-223; Williams et al.(1979) Nature 282:582-586; Herberman et al. (1979) Nature 277:221-223;and Anderson et al. (1982) J. Biol. Chem. 257(19):11301-11304.

Non-limiting examples of IFN-β polypeptides and IFN-β variantpolypeptides encompassed by the invention are set forth in Nagata et al.(1980) Nature 284:316-320; Goeddel et al. (1980) Nature 287:411-416;Yelverton et al. (1981) Nucleic Acids Res. 9:731-741; Streuli et al.(1981) Proc. Natl. Acad. Sci. U.S.A. 78:2848-2852; EP028033B1, andEP109748B1. See also U.S. Pat. Nos. 4,518,584; 4,569,908; 4,588,585;4,738,844; 4,753,795; 4,769,233; 4,793,995; 4,914,033; 4,959,314;5,545,723; and 5,814,485. These disclosures are herein incorporated byreference. These citations also provide guidance regarding residues andregions of the IFN-β polypeptide that can be altered without the loss ofbiological activity.

In one embodiment of the present invention, the IFN-β used in themethods of the invention is the mature native human IFN-β polypeptide.In another embodiment, the IFN-β is the mature IFN-β C17S polypeptide.However, the present invention encompasses other embodiments where theIFN-β is any biologically active IFN-β polypeptide or variant asdescribed elsewhere herein.

In some embodiments of the present invention, the IFN-β is recombinantlyproduced. By “recombinantly produced IFN-β” is intended IFN-β that hascomparable biological activity to native IFN-β and that has beenprepared by recombinant DNA techniques. IFN-β can be produced byculturing a host cell transformed with an expression vector comprising anucleotide sequence that encodes an IFN-β polypeptide. The host cell isone that can transcribe the nucleotide sequence and produce the desiredprotein, and can be prokaryotic (for example, E. coli) or eukaryotic(for example a yeast, insect, or mammalian cell). Examples ofrecombinant production of IFN-β are given in Mantei et al. (1982) Nature297:128; Ohno et al. (1982) Nucleic Acids Res. 10:967; Smith et al.(1983) Mol. Cell. Biol. 3:2156, and U.S. Pat. Nos. 4,462,940, 5,702,699,and 5,814,485; herein incorporated by reference.

Interferon-α

The term “IFN-α” as used herein refers to IFN-α or variants thereof,sometimes referred to as IFN-α-like polypeptides. Human alphainterferons comprise a family of about 30 protein species, encoded by atleast 14 different genes and about 16 alleles. Such IFN-α polypeptidesinclude IFN-αa, IFN-αB, IFN-αC, IFN-αD, IFN-αH, IFN-αJ, IFN-αJ1, IFN-αJ2and IFN-αK. Human IFN-α variants, which may be naturally occurring(e.g., allelic variants that occur at the IFN-α locus) or recombinantlyproduced, have amino acid sequences that are the same as, similar to, orsubstantially similar to the mature native IFN-α sequence. DNA sequencesencoding human IFN-α are also available in the art. See, for example,Goeddel et al. (1981) Nature 290:20-26 (Genbank Accession No. V00551J00209); Nagata et al. (1980) Nature 284:3126-320; Bowden et al. (1984)Gene 27:87-99 (Genbank Accession No. NM_(—)000605); and Ohara et al.(1987) FEBS Letters 211:78-82; all of which are herein incorporated byreference. Fragments of IFN-α or truncated forms of IFN-α that retaintheir activity are also encompassed. These biologically active fragmentsor truncated forms of IFN-α are generated by removing amino acidresidues from the full-length IFN-α amino acid sequence usingrecombinant DNA techniques well known in the art. IFN-α polypeptides mayfurther be glycosylated or unglycosylated.

The skilled artisan will appreciate that additional changes can beintroduced by mutation into the nucleotide sequences encoding IFN-α,thereby leading to changes in the IFN-α amino acid sequence, withoutaltering the biological activity of the interferon. Thus, an isolatednucleic acid molecule encoding an IFN-α variant having a sequence thatdiffers from human IFN-α can be created by introducing one or morenucleotide substitutions, additions, or deletions into the correspondingnucleotide sequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedIFN-α. Mutations can be introduced by standard techniques. Such variantsof IFN-α, include, for example, IFN-α-2a (Roferon-A™), IFN-α-2b (IntronA™), and IFN-αcon-1 (Infergen™). Another variant useful in the methodsof the present invention is IFN-α2a, which is disclosed in, for example,EP 43980; Meada et al. (1980) PNAS 77:7010; and Levy et al. (1981) PNAS78:6186; all of which are herein incorporated by reference. Further,variants of IFN-α can be found, for example, in U.S. Pat. No. 5,676,942,herein incorporated by reference. These citations also provide guidanceregarding residues and regions of the IFN-α polypeptide that can bealtered without the loss of biological activity.

Biologically active IFN-α variants encompassed by the invention alsoinclude IFN-α polypeptides that have covalently linked with, forexample, polyethylene glycol (PEG) or albumin. See, for example, U.S.Pat. No. 5,762,923, herein incorporated by reference.

Biologically active variants of IFN-α encompassed by the inventionshould retain IFN-α activities, particularly the ability to bind toIFN-α receptors or retain immunomodulatory, antiviral, oranit-proliferative activities. In some embodiments, the IFN-α variantretains at least about 25%, about 50%, about 75%, about 85%, about 90%,about 95%, about 98%, about 99% or more of the biological activity ofthe native IFN-α polypeptide. IFN-α variants whose activity is increasedin comparison with the activity of the native IFN-α polypeptide are alsoencompassed. The biological activity of IFN-α variants can be measuredby any method known in the art. Examples of such assays are describedabove.

In one embodiment of the present invention, the IFN-α used in themethods of the invention is the mature native human IFN-α polypeptide.However, the present invention encompasses other embodiments where theIFN-α is any biologically active IFN-α polypeptide or variant asdescribed elsewhere herein.

In some embodiments of the present invention, the IFN-α is recombinantlyproduced. By “recombinantly produced IFN-α” is intended IFN-α that hascomparable biological activity to native IFN-α and that has beenprepared by recombinant DNA techniques. IFN-α can be produced byculturing a host cell transformed with an expression vector comprising anucleotide sequence that encodes an IFN-α polypeptide. The host cell isone that can transcribe the nucleotide sequence and produce the desiredprotein, and can be prokaryotic (for example, E. coli) or eukaryotic(for example a yeast, insect, or mammalian cell). Details of the cloningof interferon-cDNA and the direct expression thereof, especially in E.coli, have in the meantime been the subject of many publications. Thus,for example, the preparation of recombinant interferons is known. See,for example, (1982) Nature 295: 503-508; (1980) Nature 284: 316-320;(1981) Nature 290: 20-26; (1980) Nucleic Acids Res. 8: 4057-4074, aswell as from European Patents Nos. 32134, 43980 and 211 148. Furtherexamples of recombinant production of IFN-α-2 are provided in Nagata etal. (1980) Nature 284:316 and European Patent 32,134. All of thesereferences are herein incorporated by reference.

Interferon-γ

The term “IFN-γ” as used herein refers to IFN-γ or variants thereof,sometimes referred to as IFN-γ-like polypeptides. IFN-γ is aglycoprotein whose mature form has 143 amino acids and a molecularweight of about 63-73 kilodaltons. The amino acid sequence of IFN-γ canbe found in, for example, U.S. Pat. No. 6,046,034, herein incorporatedby reference. Human IFN-γ variants, which may be naturally occurring(e.g., allelic variants that occur at the IFN-γ locus) or recombinantlyproduced, have amino acid sequences that are the same as, similar to, orsubstantially similar to the mature native IFN-γ sequence. DNA sequencesencoding human IFN-γ are also available in the art. See, for example,Grey et al. (1983) Proc. Natl. Acad. Sci. USA 80:5842-5846, hereinincorporated by reference. Fragments of IFN-γ or truncated forms ofIFN-γ that retain their activity are also encompassed. Thesebiologically active fragments or truncated forms of IFN-γ are generatedby removing amino acid residues from the full-length IFN-γ amino acidsequence using recombinant DNA techniques well known in the art. IFN-γpolypeptides may be glycosylated or unglycosylated.

The IFN-γ variants encompassed herein include muteins of the nativemature IFN-γ sequence. Thus, IFN-γ variants with one or more mutationsthat improve, for example, their pharmaceutical utility are alsoencompassed by the present invention.

Such IFN-γ variants are also encompassed by the present invention.Variants of IFN-γ are well known in the art. For example, U.S. Pat. No.5,770,191, herein incorporated by reference, discloses peptidescomprising the C-terminus of IFN-γ that retain the biological activityof the mature IFN-γ. Additionally, in EP 0 306870 A2, variants of humanIFN-γ were identified whose activity was significantly increased bydeleting the C-terminal 7-11 amino acids. In addition, WO 92-08737discloses a variant of recombinant human IFN-γ (IFN-γ C-10L) that hasincreased biological activity. Further variants of IFN-γ can be foundin, for example, U.S. Pat. No. 5,690,925 and U.S. Pat. No. 6,046,034both of which provide guidance as to the amino acid substitutions anddeletions that can be made in IFN-γ without losing biological activity.Each of these references is herein incorporated by reference. The aboveexamples represent non-limiting examples of IFN-γ polypeptides and IFN-γvariant polypeptides encompassed by the invention. These citations alsoprovide guidance regarding residues and regions of the IFN-γ polypeptidethat can be altered without the loss of biological activity.

Biologically active IFN-γ variants encompassed by the invention alsoinclude IFN-γ polypeptides that have covalently linked with, forexample, polyethylene glycol (PEG) or albumin.

Biologically active variants of IFN-γ encompassed by the inventionshould retain IFN-γ activities, particularly the ability to bind toIFN-γ receptors or retain immunomodulatory, antiviral, orantiproliferative activities. In some embodiments, the IFN-γ variantretains at least about 25%, about 50%, about 75%, about 85%, about 90%,about 95%, about 98%, about 99% or more of the biological activity ofthe native IFN-γ polypeptide. IFN-γ variants whose activity is increasedin comparison with the activity of the native IFN-γ polypeptide are alsoencompassed. The biological activity of IFN-γ variants can be measuredby any method known in the art. Examples of such assays are describedabove.

In one embodiment of the present invention, the IFN-γ used in themethods of the invention is the mature native human IFN-γ polypeptide.However, the present invention encompasses other embodiments where theIFN-γ is any biologically active IFN-γ polypeptide or variant asdescribed elsewhere herein.

In some embodiments of the present invention, the IFN-γ is recombinantlyproduced. By “recombinantly produced IFN-γ” is intended IFN-γ that hascomparable biological activity to native IFN-γ and that has beenprepared by recombinant DNA techniques. IFN-γ can be produced byculturing a host cell transformed with an expression vector comprising anucleotide sequence that encodes an IFN-γ polypeptide. The host cell isone that can transcribe the nucleotide sequence and produce the desiredprotein, and can be prokaryotic (for example, E. coli) or eukaryotic(for example a yeast, insect, or mammalian cell). Examples ofrecombinant production of IFN-γ are given in U.S. Pat. Nos. 6,046,034and 5,690,925; both of which are herein incorporated by reference.

Pharmaceutical Composition

Increases in the amount of cytokine in the CNS, brain, and/or spinalcord to a therapeutically effective level may be obtained viaadministration of a pharmaceutical composition including atherapeutically effective dose of this cytokine. By “therapeuticallyeffective dose” is intended a dose of cytokine that achieves the desiredgoal of increasing the amount of this cytokine in a relevant portion ofthe CNS, brain, and/or spinal cord to a therapeutically effective levelenabling a desired biological activity of the cytokine.

The invention is, in particular, directed to a composition that can beemployed for delivery of a cytokine to the CNS, brain, and/or spinalcord upon administration to tissue innervated by the olfactory and/ortrigeminal nerves. The composition can include, for example, anypharmaceutically acceptable additive, carrier, or adjuvant that issuitable for administering a cytokine to tissue innervated by theolfactory and/or trigeminal nerves. Preferably, the pharmaceuticalcomposition can be employed in diagnosis, prevention, or treatment of adisease, disorder, or injury of the CNS, brain, and/or spinal cord.Preferably, the composition includes a cytokine in combination with apharmaceutical carrier, additive, and/or adjuvant that can promote thetransfer of the cytokine within or through tissue innervated by theolfactory and/or trigeminal nerves. Alternatively, the cytokine may becombined with substances that may assist in transporting the cytokine tosites of nerve cell damage. The composition can include one or severalcytokines.

The composition typically contains a pharmaceutically acceptable carriermixed with the cytokine and other components in the pharmaceuticalcomposition. By “pharmaceutically acceptable carrier” is intended acarrier that is conventionally used in the art to facilitate thestorage, administration, and/or the healing effect of the cytokine. Acarrier may also reduce any undesirable side effects of the cytokine. Asuitable carrier should be stable, i.e., incapable of reacting withother ingredients in the formulation. It should not produce significantlocal or systemic adverse effect in recipients at the dosages andconcentrations employed for treatment. Such carriers are generally knownin the art.

Suitable carriers for this invention include those conventionally usedfor large stable macromolecules such as albumin, gelatin, collagen,polysaccharide, monosaccharides, polyvinylpyrrolidone, polylactic acid,polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate,liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran,cellulose, mannitol, sorbitol, polyethylene glycol (PEG), and the like.

Water, saline, aqueous dextrose, and glycols are preferred liquidcarriers, particularly (when isotonic) for solutions. The carrier can beselected from various oils, including those of petroleum, animal,vegetable or synthetic origin, for example, peanut oil, soybean oil,mineral oil, sesame oil, and the like. Suitable pharmaceuticalexcipients include starch, cellulose, talc, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate,sodium stearate, glycerol monostearate, sodium chloride, dried skimmilk, glycerol, propylene glycol, water, ethanol, and the like. Thecompositions can be subjected to conventional pharmaceutical expedients,such as sterilization, and can contain conventional pharmaceuticaladditives, such as preservatives, stabilizing cytokines, wetting, oremulsifying agents, salts for adjusting osmotic pressure, buffers, andthe like.

A composition formulated for intranasal delivery may optionally comprisean odorant. An odorant agent is combined with the cytokine to provide anodorliferous sensation, and/or to encourage inhalation of the intranasalpreparation to enhance delivery of the active cytokine to the olfactoryneuroepithelium. The odorliferous sensation provided by the odorantagent may be pleasant, obnoxious, or otherwise malodorous. The odorantreceptor neurons are localized to the olfactory epithelium that, inhumans, occupies only a few square centimeters in the upper part of thenasal cavity. The cilia of the olfactory neuronal dendrites whichcontain the receptors are fairly long (about 30-200 um). A 10-30 μmlayer of mucus envelops the cilia that the odorant agent must penetrateto reach the receptors. See Snyder et al. (1998) J. Biol. Chem.263:13972-13974. Use of a lipophillic odorant agent having moderate tohigh affinity for odorant binding protein (OBP) is preferred. OBP has anaffinity for small lipophillic molecules found in nasal secretions andmay act as a carrier to enhance the transport of a lipophillic odorantsubstance and cytokines to the olfactory receptor neurons. It is alsopreferred that an odorant agent is capable of associating withlipophillic additives such as liposomes and micelles within thepreparation to further enhance delivery of the cytokines by means of OBPto the olfactory neuroepithelium. OBP may also bind directly tolipophillic agents to enhance transport of the cytokines to olfactoryneural receptors.

Suitable odorants having a high affinity for OBP include terpanoids suchas cetralva and citronellol, aldehydes such as amyl clnnarmaldehyde andhexyl cinnamaldehyde, esters such as octyl isovalerate, jasmines such asC1S-jasmine and jasmal, and musk 89. Other suitable odorant agentsinclude those which may be capable of stimulating odorant-sensitiveenzymes such as aderrylate cyclase and guanylate cyclase, or which maybe capable of modifying ion channels within the olfactory system toenhance absorption of the cytokine.

Other acceptable components in the composition include, but are notlimited to, pharmaceutically acceptable agents that modify isotonicity,including water, salts, sugars, polyols, amino acids and buffers, suchas, phosphate, citrate, succinate, acetate, and other organic acids ortheir salts. Typically, the pharmaceutically acceptable carrier alsoincludes one or more stabilizers, reducing agents, anti-oxidants and/oranti-oxidant chelating agents. The use of buffers, stabilizers, reducingagents, anti-oxidants and chelating agents in the preparation of proteinbased compositions, particularly pharmaceutical compositions, is wellknown in the art. See Wang et al. (1980) J. Parent. Drug Assn.,34(6):452-462; Wang et al. (1988) J. Parent. Sci. and Tech. 42:S4-S26(Supplement); Lachman, et al. (1968) Drug and Cosmetic Industry, 102(1):36-38, 40 and 146-148; Akers, M. J. (1988) J. Parent. Sci. and Tech.,36(5):222-228; and Colowick et al. Methods in Enzymology, Vol. XXV, p.185-188.

Suitable buffers include acetate, adipate, benzoate, citrate, lactate,maleate, phosphate, tartarate, borate, tri(hydroxymethyl aminomethane),succinate, glycine, histidine, the salts of various amino acids, or thelike, or combinations thereof. See Wang (1980) supra at page 455.Suitable salts and isotonicifiers include sodium chloride, dextrose,mannitol, sucrose, trehalose, or the like. Where the carrier is aliquid, it is preferred that the carrier is hypotonic or isotonic withoral, conjunctival or dermal fluids and have a pH within the range of4.5-8.5. Where the carrier is in powdered form, it is preferred that thecarrier is also within an acceptable non-toxic pH range.

Suitable reducing agents, which maintain the reduction of reducedcysteines, include dithiothreitol (DTT also known as Cleland's reagent)or dithioerythritol at 0.01% to 0.1% wt/wt; acetylcysteine or cysteineat 0.1% to 0.5% (pH 2-3); and thioglycerol at 0.1% to 0.5% (pH 3.5 to7.0) and glutathione. See Akers (1988) supra at pages 225 to 226.Suitable antioxidants include sodium bisulfite, sodium sulfite, sodiummetabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, andascorbic acid. See Akers (1988) supra at pages 225. Suitable chelatingagents, which chelate trace metals to prevent the trace metal catalyzedoxidation of reduced cysteines, include citrate, tartarate,ethylenediaminetetraacetic acid (EDTA) in its disodium, tetrasodium, andcalcium disodium salts, and diethylenetriamine pentaacetic acid (DTPA).See, e.g. Wang (1980) supra at pages 457-458 and 460-461, and Akers(1988) supra at pages 224-227.

The composition can include one or more preservatives such as phenol,cresol, p-aminobenzoic acid, BDSA, sorbitrate, chlorhexidine,benzalkonium chloride, or the like. Suitable stabilizers includecarbohydrates such as trehalose or glycerol. The composition can includea stabilizer such as one or more of microcrystalline cellulose,magnesium stearate, mannitol, sucrose to stabilize, for example, thephysical form of the composition; and one or more of glycine, arginine,hydrolyzed collagen, or protease inhibitors to stabilize, for example,the chemical structure of the composition. Suitable suspending additivesinclude carboxymethyl cellulose, hydroxypropyl methylcellulose,hyaluronic acid, alginate, chondroitin sulfate, dextran, maltodextrin,dextran sulfate, or the like. The composition can include an emulsifiersuch as polysorbate 20, polysorbate 80, pluronic, triolein, soybean oil,lecithins, squalene and squalanes, sorbitan treioleate, or the like. Thecomposition can include an antimicrobial such as phenylethyl alcohol,phenol, cresol, benzalkonium chloride, phenoxyethanol, chlorhexidine,thimerosol, or the like. Suitable thickeners include naturalpolysaccharides such as mannans, arabinans, alginate, hyaluronic acid,dextrose, or the like; and synthetic ones like the PEG hydrogels of lowmolecular weight and aforementioned suspending cytokines.

The composition can include an adjuvant such as cetyl trimethyl ammoniumbromide, BDSA, cholate, deoxycholate, polysorbate 20 and 80, fusidicacid, or the like, and in the case of DNA delivery, preferably, acationic lipid. Suitable sugars include glycerol, threose, glucose,galactose, mannitol, and sorbitol. A suitable protein is human serumalbumin.

Preferred compositions include one or more of a solubility enhancingadditive, preferably a cyclodextrin; a hydrophilic additive, preferablya monosaccharride or oligosaccharide; an absorption promoting additive,preferably a cholate, a deoxycholate, a fusidic acid, or a chitosan; acationic surfactant, preferably a cetyl trimethyl ammonium bromide; aviscosity enhancing additive, preferably to promote residence time ofthe composition at the site of administration, preferably acarboxymethyl cellulose, a maltodextrin, an alginic acid, a hyaluronicacid, or a chondroitin sulfate; or a sustained release matrix,preferably a polyanhydride, a polyorthoester, a hydrogel, a particulateslow release depo system, preferably a polylactide co-glycolides (PLG),a depo foam, a starch microsphere, or a cellulose derived buccal system;a lipid-based carrier, preferably an emulsion, a liposome, a niosomes,or a micelles. The composition can include a bilayer destabilizingadditive, preferably a phosphatidyl ethanolamine; a fusogenic additive,preferably a cholesterol hemisuccinate.

Other preferred compositions for sublingual administration including,for example, a bioadhesive to retain the cytokine sublingually, a spray,paint, or swab applied to the tongue; retaining a slow dissolving pillor lozenge under the tongue; or the like. Other preferred compositionsfor transdermal administration include a bioadhesive to retain thecytokine on or in the skin; a spray, paint, cosmetic, or swab applied tothe skin; or the like.

These lists of carriers and additives is by no means complete and aworker skilled in the art can choose excipients from the GRAS (generallyregarded as safe) list of chemicals allowed in the pharmaceuticalpreparations and those that are currently allowed in topical andparenteral formulations.

For the purposes of this invention, the pharmaceutical compositioncomprising the cytokine can be formulated in a unit dosage and in a formsuch as a solution, suspension, or emulsion. The cytokine may beadministered to tissue innervated by the trigeminal and/or olfactoryneurons as a powder, a granule, a solution, a cream, a spray (e.g., anaerosol), a gel, an ointment, an infusion, an injection, a drop, orsustained-release composition, such as a polymer disk. For buccaladministration, the compositions can take the form of tablets orlozenges formulated in a conventional manner. For administration to theeye or other external tissues, e.g., mouth and skin, the compositionscan be applied to the infected part of the body of the patient as atopical ointment or cream. The compounds can be presented in anointment, for instance with a water-soluble ointment base, or in acream, for instance with an-oil-in water cream base. For conjunctivalapplications, the cytokine can be administered in biodegradable ornon-degradable ocular inserts. The drug may be released by matrixerosion or passively through a pore as in ethylene-vinylacetate polymerinserts. For other mucosal administrations, such as sublingual, powderdiscs may be placed under the tongue and active delivery systems may forin situ by slow hydration as in the formulation of liposomes from driedlipid mixtures or pro-liposomes.

Other preferred forms of compositions for administration include asuspension of a particulate, such as an emulsion, a liposome, an insertthat releases the cytokine slowly, and the like. The powder or granularforms of the pharmaceutical composition may be combined with a solutionand with a diluting, dispersing, or surface-active cytokine. Additionalpreferred compositions for administration include a bioadhesive toretain the cytokine at the site of administration; a spray, paint, orswab applied to the mucosa or epithelium; a slow dissolving pill orlozenge; or the like. The composition can also be in the form oflyophilized powder, which can be converted into a solution, suspension,or emulsion before administration. The pharmaceutical compositionincluding cytokine is preferably sterilized by membrane filtration andis stored in unit-dose or multi-dose containers such as sealed vials orampoules.

The method for formulating a pharmaceutical composition is generallyknown in the art. A thorough discussion of formulation and selection ofpharmaceutically acceptable carriers, stabilizers, and isomolytes can befound in Remington's Pharmaceutical Sciences (18^(th) ed.; MackPublishing Company, Eaton, Pa., 1990), herein incorporated by reference.

The cytokine of the present invention can also be formulated in asustained-release form to prolong the presence of the pharmaceuticallyactive cytokine in the treated mammal, generally for longer than oneday. Many methods of preparation of a sustained-release formulation areknown in the art and are disclosed in Remington's PharmaceuticalSciences (18^(th) ed.; Mack Publishing Company, Eaton, Pa., 1990),herein incorporated by reference.

Generally, the cytokine can be entrapped in semipermeable matrices ofsolid hydrophobic polymers. The matrices can be shaped into films ormicrocapsules. Examples of such matrices include, but are not limitedto, polyesters, copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al. (1983) Biopolymers 22:547-556),polylactides (U.S. Pat. No. 3,773,919 and EP 58,481), polylactatepolyglycolate (PLGA) such as polylactide-co-glycolide (see, for example,U.S. Pat. Nos. 4,767,628 and 5,654,008), hydrogels (see, for example,Langer et al (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982)Chem. Tech. 12:98-105), non-degradable ethylene-vinyl acetate (e.g.ethylene vinyl acetate disks and poly(ethylene-co-vinyl acetate)),degradable lactic acid-glycolic acid copolyers such as the LupronDepot™, poly-D-(−)-3-hydroxybutyric acid (EP 133,988), hyaluronic acidgels (see, for example, U.S. Pat. No. 4,636,524), alginic acidsuspensions, and the like.

Suitable microcapsules can also include hydroxymethylcellulose orgelatin-microcapsules and polymethyl methacrylate microcapsules preparedby coacervation techniques or by interfacial polymerization. See the PCTpublication WO 99/24061 entitled “Method for Producing Sustained-releaseFormulations,” wherein a protein is encapsulated in PLGA microspheres,herein incorporated by reference. In addition, microemulsions orcolloidal drug delivery systems such as liposomes and albuminmicrospheres, may also be used. See Remington's Pharmaceutical Sciences(18^(th) ed.; Mack Publishing Company Co., Eaton, Pa., 1990). Otherpreferred sustained-release compositions employ a bioadhesive to retainthe cytokine at the site of administration.

Among the optional substances that may be combined with the cytokine inthe pharmaceutical composition are lipophilic substances that canenhance absorption of the cytokine through the mucosa or epithelium ofthe nasal cavity, or along a neural, lymphatic, or perivascular pathwayto damaged nerve cells in the CNS. The cytokine may be mixed with alipophilic adjuvant alone or in combination with a carrier, or may becombined with one or several types of micelle or liposome substances.Among the preferred lipophilic substances are cationic liposomesincluded of one or more of the following: phosphatidyl choline,lipofectin, DOTAP, a lipid-peptoid conjugate, a synthetic phospholipidsuch as phosphatidyl lysine, or the like. These liposomes may includeother lipophilic substances such as gangliosides and phosphatidylserine(PS). Also preferred are micellar additives such as GM-1 gangliosidesand phosphatidylserine (PS), which may be combined with the cytokineeither alone or in combination. GM-1 ganglioside can be included at 1-10mole percent in any liposomal compositions or in higher amounts inmicellar structures. Protein cytokines can be either encapsulated inparticulate structures or incorporated as part of the hydrophobicportion of the structure depending on the hydrophobicity of the activecytokine.

One preferred liposomal formulation employs Depofoam. A cytokine can beencapsulated in multivesicular liposomes, as disclosed in the WOpublication 99/12522 entitled “High and Low Load Formulations of IGF-Iin Multivesicular Liposomes,” herein incorporated by reference. The meanresidence time of cytokine at the site of administration can beprolonged with a Depofoam composition.

Administering the Cytokine

According to this embodiment of the invention, the total amount ofcytokine administered per dose should be in a range sufficient todelivery a biologically relevant amount of the cytokine (i.e., an amountsufficient to produce a therapeutical effect). The pharmaceuticalcomposition having a unit dose of cytokine can be in the form ofsolution, suspension, emulsion, or a sustained-release formulation. Thetotal volume of one dose of the pharmaceutical composition can rangefrom about 10 μl to about 100 μl, for example, for nasal administration.It is apparent that the suitable volume can vary with factors such asthe size of the tissue to which the cytokine is administered and thesolubility of the components in the composition.

It is recognized that the total amount of cytokine administered as aunit dose to a particular tissue will depend upon the type ofpharmaceutical composition being administered, that is whether thecomposition is in the form of, for example, a solution, a suspension, anemulsion, or a sustained-release formulation. For example, where thepharmaceutical composition comprising a therapeutically effective amountof cytokine is a sustained-release formulation, cytokine is administeredat a higher concentration. Needle-free subcutaneous administration to anextranasal tissue innervated by the trigeminal nerve may be accomplishedby use of a device which employs a supersonic gas jet as a power sourceto accelerate an agent that is formulated as a powder or a microparticleinto the skin. The characteristics of such a delivery method will bedetermined by the properties of the particle, the formulation of theagent and the gas dynamics of the delivery device. Similarly, thesubcutaneous delivery of an aqueous composition can be accomplished in aneedle-free manner by employing a gas-spring powered hand held device toproduce a high force jet of fluid capable of penetrating the skin.Alternatively, a skin-patch formulated to mediate a sustained release ofa composition can be employed for the transdermal delivery of aneuroregulatory agent to a tissue innervated by the trigeminal nerve.Where the pharmaceutical composition comprises a therapeuticallyeffective amount of an agent, or a combination of agents, in asustained-release formulation, the agent(s) is/are administered at ahigher concentration.

It should be apparent to a person skilled in the art that variations maybe acceptable with respect to the therapeutically effective dose andfrequency of the administration a cytokine in this embodiment of theinvention. The amount of the cytokine administered will be inverselycorrelated with the frequency of administration. Hence, an increase inthe concentration of cytokine in a single administered dose, or anincrease in the mean residence time in the case of a sustained-releaseform of cytokine, generally will be coupled with a decrease in thefrequency of administration.

In the practice of the present invention, additional factors should betaken into consideration when determining the therapeutically effectivedose of cytokine and frequency of its administration. Such factorsinclude, for example, the size of the tissue, the area of the surface ofthe tissue, the severity of the disease or disorder, and the age,height, weight, health, and physical condition of the individual to betreated. Generally, a higher dosage is preferred if the tissue is largeror the disease or disorder is more severe.

Some minor degree of experimentation may be required to determine themost effective dose and frequency of dose administration, this beingwell within the capability of one skilled in the art once apprised ofthe present disclosure.

For the treatment of a disorder of the CNS in a human, includingneurologic, viral, proliferative or immunomodulatory disorders, atherapeutically effective amount or dose of a cytokine is about 0.14nmol/kg of brain weight to about 138 nmol/kg brain weight and about 0.14nmol/kg of brain weight to about 69 nmol/kg of brain weight. In someregimens, therapeutically effective doses for administration of acytokine include about 0.13, 0.2, 0.4, 0.6, 0.8, 1.0, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, or 140-nmoles per kg of brainweight. For the treatment of a disorder of the CNS in a human, includingneurologic, viral, proliferative or immunomodulatory disorders, thetherapeutically effective amount or dose of IFN-β or biologically activevariant thereof is about 0.14 nmol/kg of brain weight to about 138nmol/kg of brain weight and about 0.14 nmol/kg of brain weight to about69 nmol/kg of brain weight. In some regimens, therapeutically effectivedoses for administration of IFN-β include about 0.13, 0.2, 0.4, 0.6,0.8, 1.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140nmoles per kg of brain weight.

It is further recognized that the therapeutically effective amount ordose of a cytokine to a human may be lower when the cytokine isadministered via the nasal lymphatics to various tissues of the head andneck for the treatment or prevention of disorders or diseasescharacterized by immune and inflammatory responses (i.e., diseasesresulting in acute or chronic inflammation and/or infiltration bylymphocytes). In these embodiments, while the cytokine can beadministered in the dosage range provided above, the cytokine may alsobe administered from about 0.02 to about 138 pmol/kg of brain weight.Alternatively, the cytokine may be administered from about 0.02, 0.03,0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 pmol per kg of brainweight. Similarly, when the cytokine is IFN-β, the dosage range may alsobe from about 0.02 to about 138 pmol/kg of brain weight. Alternatively,the cytokine may be administered from about 0.02, 0.03, 0.08, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, or 140 pmol per kg of brain weight.

These doses depend on factors including the efficiency with whichcytokine IFN-β is transported to the CNS or lymphatic system. A largertotal dose can be delivered by multiple administrations of the agent.

Intermittent Dosing

In another embodiment of the invention, the pharmaceutical compositioncomprising the therapeutically effective dose of cytokine isadministered intermittently. By “intermittent administration” isintended administration of a therapeutically effective dose of cytokine,followed by a time period of discontinuance, which is then followed byanother administration of a therapeutically effective dose, and soforth. Administration of the therapeutically effective dose may beachieved in a continuous manner, as for example with a sustained-releaseformulation, or it may be achieved according to a desired daily dosageregimen, as for example with one, two, three or more administrations perday. By “time period of discontinuance” is intended a discontinuing ofthe continuous sustained-released or daily administration of cytokine.The time period of discontinuance may be longer or shorter than theperiod of continuous sustained-release or daily administration. Duringthe time period of discontinuance, the cytokine level in the relevanttissue is substantially below the maximum level obtained during thetreatment. The preferred length of the discontinuance period depends onthe concentration of the effective dose and the form of cytokine used.The discontinuance period can be at least 2 days, preferably is at least4 days, more preferably is at least 1 week and generally does not exceeda period of 4 weeks. When a sustained-release formulation is used, thediscontinuance period must be extended to account for the greaterresidence time of cytokine at the site of injury. Alternatively, thefrequency of administration of the effective dose of thesustained-release formulation can be decreased accordingly. Anintermittent schedule of administration of cytokine can continue untilthe desired therapeutic effect, and ultimately treatment of the diseaseor disorder, is achieved.

In yet another embodiment, intermittent administration of thetherapeutically effective dose of cytokine is cyclic. By “cyclic” isintended intermittent administration accompanied by breaks in theadministration, with cycles ranging from about 1 month to about 2, 3, 4,5, or 6 months. For example, the administration schedule might beintermittent administration of the effective dose of cytokine, wherein asingle short-term dose is given once per week for 4 weeks, followed by abreak in intermittent administration for a period of 3 months, followedby intermittent administration by administration of a single short-termdose given once per week for 4 weeks, followed by a break inintermittent administration for a period of 3 months, and so forth. Asanother example, a single short-term dose may be given once per week for2 weeks, followed by a break in intermittent administration for a periodof 1 month, followed by a single short-term dose given once per week for2 weeks, followed by a break in intermittent administration for a periodof 1 month, and so forth. A cyclic intermittent schedule ofadministration of cytokine to subject may continue until the desiredtherapeutic effect, and ultimately treatment of the disorder or disease,is achieved.

Neuronal Transport

One embodiment of the present method includes administration of thecytokine to the subject in a manner such that the cytokine istransported to the lymphatic system, the lacrimal gland, CNS, brain,and/or spinal cord along a neural pathway. A neural pathway includestransport within or along a neuron, through or by way of lymphaticsrunning with a neuron, through or by way of a perivascular space of ablood vessel running with a neuron or neural pathway, through or by wayof an adventitia of a blood vessel running with a neuron or neuralpathway, or through an hemangiolymphatic system. The invention preferstransport of a cytokine by way of a neural pathway, rather than throughthe circulatory system, so that cytokines that are unable to, or onlypoorly, cross the blood-brain barrier from the bloodstream into thebrain can be delivered to the lymphatic system, CNS, brain, and/orspinal cord. The cytokine, once past the blood-brain barrier and in theCNS, can then be delivered to various areas of the brain or spinal cordthrough lymphatic channels, through a perivascular space, or transportthrough or along neurons. In one embodiment, the cytokine preferablyaccumulates in areas having the greatest density of receptor or bindingsites for that cytokine.

Use of a neural pathway to transport a cytokine to the lymphatic system,lacrimal gland, brain, spinal cord, or other components of the centralnervous system obviates the obstacle presented by the blood-brainbarrier so that medications that cannot normally cross that barrier, canbe delivered directly to the brain, cerebellum, brain stem, or spinalcord. Although the cytokine that is administered may be absorbed intothe bloodstream as well as the neural pathway, the cytokine preferablyprovides minimal effects systemically. In addition, the invention canprovide for delivery of a more concentrated level of the cytokine toneural cells since the cytokine does not become diluted in fluidspresent in the bloodstream. As such, the invention provides an improvedmethod for delivering a cytokine to the lymphatic system, CNS, brain,and/or spinal cord.

The Olfactory Neural Pathway

One embodiment of the present method includes delivery of the cytokineto the subject in a manner such that the cytokine is transported intothe CNS, brain, and/or spinal cord along an olfactory neural pathway.Typically, such an embodiment includes administering the cytokine totissue innervated by the olfactory nerve and inside the nasal cavity.The olfactory neural pathway innervates primarily the olfactoryepithelium in the upper third of the nasal cavity, as described above.Application of the cytokine to a tissue innervated by the olfactorynerve can deliver the cytokine to damaged neurons or cells of the CNS,brain, and/or spinal cord. Olfactory neurons innervate this tissue andcan provide a direct connection to the CNS, brain, and/or spinal corddue, it is believed, to their role in olfaction.

Delivery through the olfactory neural pathway can employ lymphatics thattravel with the olfactory nerve to the various brain areas and fromthere into dural lymphatics associated with portions of the CNS, such asthe spinal cord. Transport along the olfactory nerve can also delivercytokines to an olfactory bulb. A perivascular pathway and/or ahemangiolymphatic pathway, such as lymphatic channels running within theadventitia of cerebral blood vessels, can provide an additionalmechanism for transport of therapeutic cytokines to the brain and spinalcord from tissue innervated by the olfactory nerve.

A cytokine can be administered to the olfactory nerve, for example,through the olfactory epithelium. Such administration can employextracellular or intracellular (e.g., transneuronal) anterograde andretrograde transport of the cytokine entering through the olfactorynerves to the brain and its meninges, to the brain stem, or to thespinal cord. Once the cytokine is dispensed into or onto tissueinnervated by the olfactory nerve, the cytokine may transport throughthe tissue and travel along olfactory neurons into areas of the CNSincluding the brain stem, cerebellum, spinal cord, olfactory bulb, andcortical and subcortical structures.

Delivery through the olfactory neural pathway can employ movement of acytokine into or across mucosa or epithelium into the olfactory nerve orinto a lymphatic, a blood vessel perivascular space, a blood vesseladventitia, or a blood vessel lymphatic that travels with the olfactorynerve to the brain and from there into meningial lymphatics associatedwith portions of the CNS such as the spinal cord. Blood vessellymphatics include lymphatic channels that are around the blood vesselson the outside of the blood vessels. This also is referred to as thehemangiolymphatic system. Introduction of a cytokine into the bloodvessel lymphatics does not necessarily introduce the cytokine into theblood.

The Trigeminal Neural Pathway

One embodiment of the present method includes delivery of the cytokineto the subject in a manner such that the cytokine is transported intothe CNS, brain, and/or spinal cord along a trigeminal neural pathway.Typically, such an embodiment includes administering the cytokine totissue innervated by the trigeminal nerve including inside and outsidethe nasal cavity. The trigeminal neural pathway innervates varioustissues of the head and face, as described above. In particular, thetrigeminal nerve innervates the nasal, sinusoidal, oral and conjunctivalmucosa or epithelium, and the skin of the face. Application of thecytokine to a tissue innervated by the trigeminal nerve can deliver thecytokine to damaged neurons or cells of the CNS, brain, and/or spinalcord to cells of the lymphatic system. Trigeminal neurons innervatethese tissues and can provide a direct connection to the CNS, brain,and/or spinal cord due, it is believed, to their role in the commonchemical sense including mechanical sensation, thermal sensation andnociception (for example detection of hot spices and of noxiouschemicals).

Delivery through the trigeminal neural pathway can employ lymphaticsthat travel with the trigeminal nerve to the pons and other brain areasand from there into dural lymphatics associated with portions of theCNS, such as the spinal cord. Transport along the trigeminal nerve canalso deliver cytokines to an olfactory bulb. A perivascular pathwayand/or a hemangiolymphatic pathway, such as lymphatic channels runningwithin the adventitia of cerebral blood vessels, can provide anadditional mechanism for transport of therapeutic cytokines to thespinal cord from tissue innervated by the trigeminal nerve.

The trigeminal nerve includes large diameter axons, which mediatemechanical sensation, e.g., touch, and small diameter axons, whichmediate pain and thermal sensation, both of whose cell bodies arelocated in the semilunar (or trigeminal) ganglion or the mesencephalictrigeminal nucleus in the midbrain. Certain portions of the trigeminalnerve extend into the nasal cavity, oral and conjunctival mucosa and/orepithelium. Other portions of the trigeminal nerve extend into the skinof the face, forehead, upper eyelid, lower eyelid, dorsum of the nose,side of the nose, upper lip, cheek, chin, scalp and teeth. Individualfibers of the trigeminal nerve collect into a large bundle, travelunderneath the brain and enter the ventral aspect of the pons. Acytokine can be administered to the trigeminal nerve, for example,through the nasal cavity's, oral, lingual, and/or conjunctival mucosaand/or epithelium; or through the skin of the face, forehead, uppereyelid, lower eyelid, dorsum of the nose, side of the nose, upper lip,cheek, chin, scalp and teeth. Such administration can employextracellular or intracellular (e.g., transneuronal) anterograde andretrograde transport of the cytokine entering through the trigeminalnerves to the brain and its meninges, to the brain stem, or to thespinal cord. Once the cytokine is dispensed into or onto tissueinnervated by the trigeminal nerve, the cytokine may transport throughthe tissue and travel along trigeminal neurons into areas of the CNSincluding the brain stem, cerebellum, spinal cord, olfactory bulb, andcortical and subcortical structures.

Delivery through the trigeminal neural pathway can employ movement of acytokine across skin, mucosa, or epithelium into the trigeminal nerve orinto a lymphatic, a blood vessel perivascular space, a blood vesseladventitia, or a blood vessel lymphatic that travels with the trigeminalnerve to the pons and from there into meningial lymphatics associatedwith portions of the CNS such as the spinal cord. Blood vessellymphatics include lymphatic channels that are around the blood vesselson the outside of the blood vessels. This also is referred to as thehemangiolymphatic system. Introduction of a cytokine into the bloodvessel lymphatics does not necessarily introduce the cytokine into theblood.

Neural Pathways and Nasal Administration

In one embodiment, the method of the invention can employ delivery by aneural pathway, e.g., a trigeminal or olfactory neural pathway, afteradministration to the nasal cavity. Upon administration to the nasalcavity, delivery via the trigeminal neural pathway may employ movementof a cytokine through the nasal mucosa and/or epithelium to reach atrigeminal nerve or a perivascular and/or lymphatic channel that travelswith the nerve. Upon administration to the nasal cavity, delivery viathe olfactory neural pathway may employ movement of a cytokine throughthe nasal mucosa and/or epithelium to reach the olfactory nerve or aperivascular and/or lymphatic channel that travels with the nerve.

For example, the cytokine can be administered to the nasal cavity in amanner that employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport into and along the trigeminaland/or olfactory nerves to reach the brain, the brain stem, or thespinal cord. Once the cytokine is dispensed into or onto nasal mucosaand/or epithelium innervated by the trigeminal and/or olfactory nerve,the cytokine may transport through the nasal mucosa and/or epitheliumand travel along trigeminal and/or olfactory neurons into areas of theCNS including the brain stem, cerebellum, spinal cord, olfactory bulb,and cortical and subcortical structures. Alternatively, administrationto the nasal cavity can result in delivery of a cytokine into a bloodvessel perivascular space or a lymphatic that travels with thetrigeminal and/or olfactory nerve to the pons, olfactory bulb, and otherbrain areas, and from there into meningeal lymphatics associated withportions of the CNS such as the spinal cord. Transport along thetrigeminal and/or olfactory nerve may also deliver cytokinesadministered to the nasal cavity to the olfactory bulb, midbrain,diencephalon, medulla, and cerebellum. A cytokine administered to thenasal cavity can enter the ventral dura of the brain and travel inlymphatic channels within the dura.

In addition, the method of the invention can be carried out in a waythat employs a perivascular pathway and/or an hemangiolymphatic pathway,such as a lymphatic channel running within the adventitia of a cerebralblood vessel, to provide an additional mechanism for transport ofcytokine to the spinal cord from the nasal mucosa and/or epithelium. Acytokine transported by the hemangiolymphatic pathway does notnecessarily enter the circulation. Blood vessel lymphatics associatedwith the circle of Willis as well as blood vessels following thetrigeminal and/or olfactory nerve can also be involved in the transportof the cytokine.

Administration to the nasal cavity employing a neural pathway candeliver a cytokine to the lymphatic system, brain stem, cerebellum,spinal cord, and cortical and subcortical structures. The cytokine alonemay facilitate this movement into the CNS, brain, and/or spinal cord.Alternatively, the carrier or other transfer-promoting factors mayassist in the transport of the cytokine into and along the trigeminaland/or olfactory neural pathway. Administration to the nasal cavity of atherapeutic cytokine can bypass the blood-brain barrier through atransport system from the nasal mucosa and/or epithelium to the brainand spinal cord.

Neural Pathways and Transdermal Administration

In one embodiment, the method of the invention can employ delivery by aneural pathway, e.g., a trigeminal neural pathway, after transdermaladministration. Upon transdermal administration, delivery via thetrigeminal neural pathway may employ movement of a cytokine through theskin to reach a trigeminal nerve or a perivascular and/or lymphaticchannel that travels with the nerve.

For example, the cytokine can be administered transdermally in a mannerthat employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport into and along the trigeminalnerves to reach the brain, the brain stem, or the spinal cord. Once thecytokine is dispensed into or onto skin innervated by the trigeminalnerve, the cytokine may transport through the skin and travel alongtrigeminal neurons into areas of the CNS including the brain stem,cerebellum, spinal cord, olfactory bulb, and cortical and subcorticalstructures. Alternatively, transdermal administration can result indelivery of a cytokine into a blood vessel perivascular space or alymphatic that travels with the trigeminal nerve to the pons, olfactorybulb, and other brain areas, and from there into meningeal lymphaticsassociated with portions of the CNS such as the spinal cord. Transportalong the trigeminal nerve may also deliver transdermally administeredcytokines to the olfactory bulb, midbrain, diencephalon, medulla andcerebellum. The ethmoidal branch of the trigeminal nerve enters thecribriform region. An transdermally administered cytokine can enter theventral dura of the brain and travel in lymphatic channels within thedura.

In addition, the method of the invention can be carried out in a waythat employs a perivascular pathway and/or an hemangiolymphatic pathway,such as a lymphatic channel running within the adventitia of a cerebralblood vessel, to provide an additional mechanism for transport ofcytokine to the spinal cord from the skin. A cytokine transported by thehemangiolymphatic pathway does not necessarily enter the circulation.Blood vessel lymphatics associated with the circle of Willis as well asblood vessels following the trigeminal nerve can also be involved in thetransport of the cytokine.

Transdermal administration employing a neural pathway can deliver acytokine to the brain stem, cerebellum, spinal cord and cortical andsubcortical structures. The cytokine alone may facilitate this movementinto the CNS, brain, and/or spinal cord. Alternatively, the carrier orother transfer-promoting factors may assist in the transport of thecytokine into and along the trigeminal neural pathway. Transdermaladministration of a therapeutic cytokine can bypass the blood-brainbarrier through a transport system from the skin to the brain and spinalcord.

Neural Pathways and Sublingual Administration

In another embodiment, the method of the invention can employ deliveryby a neural pathway, e.g., a trigeminal neural pathway, after sublingualadministration. Upon sublingual administration, delivery via thetrigeminal neural pathway may employ movement of a cytokine from underthe tongue and across the lingual epithelium to reach a trigeminal nerveor a perivascular or lymphatic channel that travels with the nerve.

For example, the cytokine can be administered sublingually in a mannerthat employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport through the oral mucosa and theninto and along the trigeminal nerves to reach the brain, the brain stem,or the spinal cord. Once the cytokine is administered sublingually, thecytokine may transport through the oral mucosa by means of theperipheral processes of trigeminal neurons into areas of the CNSincluding the brain stem, spinal cord and cortical and subcorticalstructures. Alternatively, sublingual administration can result indelivery of a cytokine into lymphatics that travel with the trigeminalnerve to the pons and other brain areas and from there into meningeallymphatics associated with portions of the CNS such as the spinal cord.Transport along the trigeminal nerve may also deliver sublinguallyadministered cytokines to the olfactory bulbs, midbrain, diencephalon,medulla and cerebellum. The ethmoidal branch of the trigeminal nerveenters the cribriform region. A sublingually administered cytokine canenter the ventral dura of the brain and travel in lymphatic channelswithin the dura

In addition, the method of the invention can be carried out in a waythat employs an hemangiolymphatic pathway, such as a lymphatic channelrunning within the adventitia of a cerebral blood vessel, to provide anadditional mechanism for transport of a cytokine to the spinal cord fromthe oral submucosa. A cytokine transported by the hemangiolymphaticpathway does not necessarily enter the circulation. Blood vessellymphatics associated with the circle of Willis as well as blood vesselsfollowing the trigeminal nerve can also be involved in the transport ofthe cytokine.

Sublingual administration employing a neural pathway can deliver acytokine to the brain stem, cerebellum, spinal cord and cortical andsubcortical structures. The cytokine alone may facilitate this movementinto the CNS, brain, and/or spinal cord. Alternatively, the carrier orother transfer-promoting factors may assist in the transport of thecytokine into and along the trigeminal neural pathway. Sublingualadministration of a therapeutic cytokine can bypass the blood-brainbarrier through a transport system from the oral mucosa to the brain andspinal cord.

Neural Pathways and Conjunctival Administration

In another embodiment, the method of the invention can employ deliveryby a neural pathway, e.g. a trigeminal neural pathway, afterconjunctival administration. Upon conjunctival administration, deliveryvia the trigeminal neural pathway may employ movement of a cytokine fromthe conjunctiva through the conjunctival epithelium to reach thetrigeminal nerves or lymphatic channels that travel with the nerve.

For example, the cytokine can be administered conjunctivally in a mannerthat employs extracellular or intracellular (e.g., transneuronal)anterograde and retrograde transport through the conjunctival mucosa andthen into and along the trigeminal nerves to reach the brain, the brainstem, or the spinal cord. Once the cytokine is administeredconjunctivally, the cytokine may transport through the conjunctivalmucosa by means of the peripheral processes of trigeminal neurons intoareas of the CNS including the brain stem, spinal cord and cortical andsubcortical structures. Alternatively, conjunctival administration canresult in delivery of a cytokine into lymphatics that travel with thetrigeminal nerve to the pons and other brain areas and from there intomeningeal lymphatics associated with portions of the CNS such as thespinal cord. Transport along the trigeminal nerve may also deliverconjunctivally administered cytokines to the olfactory bulbs, midbrain,diencephalon, medulla and cerebellum. The ethmoidal branch of thetrigeminal nerve enters the cribriform region. An conjunctivallyadministered cytokine can enter the ventral dura of the brain and travelin lymphatic channels within the dura.

In addition, the method of the invention can be carried out in a waythat employs an hemangiolymphatic pathway, such as a lymphatic channelrunning within the adventitia of cerebral blood vessel, to provide anadditional mechanism for transport of a cytokine to the spinal cord fromthe conjunctival submucosa. A cytokine transported by thehemangiolymphatic pathway does not necessarily enter the circulation.Blood vessel lymphatics associated with the circle of Willis as well asblood vessels following the trigeminal nerve can also be involved in thetransport of the cytokine.

Conjunctival administration employing a neural pathway can deliver acytokine to the brain stem, cerebellum, spinal cord and cortical andsubcortical structures. The cytokine alone may facilitate this movementinto the CNS, brain, and/or spinal cord. Alternatively, the carrier orother transfer-promoting factors may assist in the transport of thecytokine into and along the trigeminal neural pathway. Conjunctivaladministration of a therapeutic cytokine can bypass the blood-brainbarrier through a transport system from the conjunctival mucosa to thebrain and spinal cord.

Articles and Methods of Manufacture

The present invention also includes an article of manufacture providinga cytokine for administration to the CNS, brain, and/or spinal cord. Thearticle of manufacture can include a vial or other container thatcontains a composition suitable for the present method together with anycarrier, either dried or in liquid form. The article of manufacturefurther includes instructions in the form of a label on the containerand/or in the form of an insert included in a box in which the containeris packaged, for the carrying out the method of the invention. Theinstructions can also be printed on the box in which the vial ispackaged. The instructions contain information such as sufficient dosageand administration information so as to allow the subject or a worker inthe field to administer the cytokine. It is anticipated that a worker inthe field encompasses any doctor, nurse, technician, spouse, or othercare-giver that might administer the cytokine. The cytokine can also beself-administered by the subject.

According to the invention, a cytokine can be used for manufacturing acytokine composition or medicament suitable for intranasal,conjunctival, transdermal, and/or sublingual administration. Forexample, a liquid or solid composition can be manufactured in severalways, using conventional techniques. A liquid composition can bemanufactured by dissolving a cytokine in a suitable solvent, such aswater, at an appropriate pH, including buffers or other excipients, forexample to form a solution described herein above.

Disorders of the Central Nervous System

In one embodiment, the present method can be employed to delivercytokines, particularly IFN-β, to the brain for diagnosis, treatment orprevention of disorders or diseases of the CNS, brain, and/or spinalcord. IFN-β increases the astrocytic production of nerve growth factor(NGF) (Boutros et al. (1997) Journal of Neurochemistry 69:939-946) andIFN-β sustains neuronal growth in cell culture (Plioplys et al. (1995)Neuroimmunodulation 2:131-135). IFN-β has therefore been associated withneurotrophic activity, hence, the methods of the present invention canbe used for the delivery of a cytokine to the CNS to treat or preventdisorders or diseases of the CNS, brain, and/or spinal cord.

Disorders of the CNS, brain and/or spinal cord can be neurologic orpsychiatric disorders, and include, for example, brain diseases such asAlzheimer's disease, Parkinson's disease, Lewy body dementia, multiplesclerosis, epilepsy, cerebellar ataxia, progressive supranuclear palsy,amyotrophic lateral sclerosis, affective disorders, anxiety disorders,obsessive compulsive disorders, personality disorders, attention deficitdisorder, attention deficit hyperactivity disorder, Tourette Syndrome,Tay Sachs, Nieman Pick, and other lipid storage and genetic braindiseases and/or schizophrenia. The method can also be employed insubjects suffering from or at risk for nerve damage from cerebrovasculardisorders such as stroke in the brain or spinal cord, from CNSinfections including meningitis and HIV, from tumors of the brain andspinal cord, or from a prior disease. The method can also be employed todeliver cytokines to counter CNS disorders resulting from ordinary aging(e.g., anosmia or loss of the general chemical sense), brain injury, orspinal cord injury.

Multiple sclerosis is a preferred disease or disorder of the CNS, brain,and/or spinal cord. Despite its possible presence in the periphery,multiple sclerosis is a disease of the CNS. Accordingly, multiplesclerosis may be targeted more efficiently by a method deliveringinterferons to the CNS, brain and/or spinal cord.

Another preferred disease of the CNS, brain, and/or spinal cord ismeningitis.

An “effective amount” of a cytokine is an amount sufficient to prevent,treat, reduce and/or ameliorate the symptoms and/or underlying causes ofany of the above disorders or diseases discussed herein. In someinstances, an “effective amount” is sufficient to eliminate the symptomsof those diseases and, perhaps, overcome the disease itself. In thecontext of the present invention, the terms “treat” and “therapy” andthe like refer to alleviate, slow the progression, prophylaxis,attenuation or cure of existing disease. Prevent, as used herein, refersto delaying, slowing, inhibiting, reducing or ameliorating the onset ofthe CNS or brain diseases or disorders. It is preferred that asufficient quantity of the cytokine be applied in non-toxic levels inorder to provide an effective level of activity within the CNS toprevent or treat the disease. The method of the present invention may beused with any mammal. Exemplary mammals include, but are not limited torats, cats, dogs, horses, cows, sheep, pigs, and more preferably humans.

Further Embodiments

Modulation of Immune and Inflammatory Responses

The method of cytokine administration provided by the present inventionallows for the directed administration of the cytokine to the nasallymphatic system. Following entry of the cytokine into the nasallymphatics, the cytokine can be distributed throughout the lymphatics ofthe head and neck region. Hence, the method of the present invention canbe employed to deliver cytokines to the lymphatic system including, forexample, the deep and superficial cervical nodes, and to various tissuesof the head and neck for the treatment or prevention of disorders ordiseases characterized by immune and inflammatory responses (i.e.,diseases resulting in acute or chronic inflammation and/or infiltrationby lymphocytes). As such the present invention provides a method tomodulate the immune response. By modulate is intended any up or downregulation of the immune or inflammatory response (i.e., influencingsystemic immune function, antigen presentation, cytokine production, andentry of leukocytes into the CNS).

Of particular interest in the methods of the invention is theadministration of IFN-β. IFN-β, like many of the interferons, reportedlyserves as an immunomodulator on a number of target cells (Hall et al.(1997) J. Neuroimmunol. 72:11-19). For instance, IFN-β appears to exertantiproliferative action on macrophages, counteract “the mitogenicstimulus of certain cytokines”, augment natural killer cell activity toinduce an increase in the production of cytotoxic T lymphocytes, and acton large, granular lymphocytes to increase killer cell activity.Additionally, IFN-β augments the proliferation of B cells and thesecretion of IgM, IgG, and IgA. It has been shown to upregulate class IMHC expression to produce an increase in the presentation of class Irestricted antigen CD8 cells (Hall et al. (1997) J. Neuroimmunol.72:11-19). Conversely, IFN-β exerts an inhibitory effect on theupregulation of class II surface expression Hence, the immunomodulatoryactivities of IFN-β include, for example, influencing systemic immunefunction, antigen presentation, cytokine production, and entry ofleukocytes into the CNS (Yong et al. (1998) Neurology 51:582-689).Direct delivery of the cytokine to the lymphatics of the head and neckusing the administration methods of the present invention allows thecytokine to modulate the immune response, i.e., influence chronic andacute inflammation, wound healing, and the autoimmune response; modulatethe function by lymphocytes (reduce lymphocyte infiltration of theinjured tissue); etc.

Given the immunomodulatory role of cytokines, the present invention canbe employed to deliver cytokines, preferably IFN-β, to various tissuesof the head and neck for the treatment and/or prevention of diseases ordisorders characterized by immune and inflammatory responses. Disordersor diseases of particular interest include Multiple Sclerosis (MS),meningitis, and Primary Sjogren's Syndrome.

MS presents in the white matter of the CNS and spinal cord as a numberof sclerotic lesions or plaques (Prineas (1985) Demyelinating Diseases,Elsvevier: Amsterdam; Raine (1983) Multiple Sclerosis, Williams andWilkins: Baltimore; Raine et al. (1988) J. Neuroimmunol. 20:189-201; andMartin (1997) J. Neural Transmission (Suppl) 49:53-67). Thecharacteristic MS lesion is inflamed, exhibits axonal demyelination,axonal degeneration, and is found around small venules. Thesecharacteristics typically evolve early in plaque development and arehypothesized to occur as a result of a breakdown in the blood-brainbarrier (BBB). As a consequence of BBB breakdown, infiltrates consistingof various lymphocytes and macrophages enter the brain. The infiltratescause a decrease in inflammation while increasing the presence of glialscar tissue, and elicit incomplete remyelination (Martin (1997) J.Neural Transmission (Suppl) 49:53-67). Further, it is hypothesized thatthis apparent immunologic attack targets not only the myelin sheath, butalso the oligodendrocytes imperative to CNS myelin production. Cytokinesare known to effectively reduce the symptoms of MS. For example,interferon-β (IFN-β) has received interest as a treatment forrelapsing-remitting MS. In addition, interest has also developed in theuse of interferon-τ as an effective treatment in autoimmune diseases,such as MS. See, for example, U.S. Pat. No. 6,060,450, hereinincorporated by reference.

The immunomodulating activity of IFN-β influences the clinical symptomsof MS. Hence, IFN-β can be administered according to the methods of thepresent invention to treat MS. While the present invention is not boundby the mechanism of IFN-β action, the central nervous system damage thatensues in MS patients is believed to be due to the delayed-typehypersensitivity response. This is a cell-mediated response. First, Tcells are activated by antigens and conveyed to the lymphoid organ(activation). The lymphoid organ then activates these T cells whilecontinuing to recruit more T cells to its site (recruitment). Theactivated lymphocytes proliferate and return to circulation (expansion).Once returned to circulation, the activated lymphocytes migrate throughthe blood stream, crossing endothelial cells lining the capillaries(migration). These migrating lymphocytes and macrophages target, and areattracted to the area of inflammation (attraction). Resulting from thisattraction, other lymphocytes continue to the area of inflammation andtissue is destroyed (tissue destruction). Subsequently, the acuteresponse is suppressed (via tissue destruction), and repair of the areaof inflammation, which is quite limited in MS, may commence (repair)(Kelley (1996) J. of Neuroscience Nursing 28:114-120). Therefore, themigration of activated lymphocytes from the blood initiates the immuneresponse, thereby allowing BBB penetration of activated lymphocytes.

Evidence suggests that the immunomodulatory activity of IFN-β inhibitsIFN-γ upregulation by inhibiting the expansion stage of the delayed-typehypersensitivity response and thereby influences the clinical symptomsof MS. Particularly, the reduction of myelin damage appears to occur asa result of two hypothesized mechanisms of IFN-β action: (1) inhibitionof IFN->induced macrophage activation, and (2) inhibition of monocytoticTNF release (Kelly (1996) J. Neuroscience Nursing 28:114-120). Potentialsites of IFN-β action construed by these hypotheses involve systemicimmune function, antigen presentation, cytokine production, and entry ofleukocytes into the CNS (Yong et al. (1998) Neurology 51:682-689). Eachof these sites has been elaborated in human and animal experiments ofMS.

An “effective amount” of a cytokine to treat MS using the administrationmethods of the present invention will be sufficient to reduce or lessenthe clinical symptoms of MS. For instance, experimental allergicencephalomyelitis (EAE) is commonly used as an animal model of MS. Atherapeutically effect amount of a cytokine delivered by the methods ofthe present invention will be such as to improve the clinical symptomsof EAE in the experimental animal (i.e., rats or mice). EAE in rats isscored on a scale of 0-4: 0, clinically normal; 1, flaccid tailparalysis; 2, hind limb weakness; 3, hind limb paralysis; 4, front andhind limb affected. An effective amount of cytokine delivered by themethods of the present invention will be effective if there is at leasta 30%, 40%, 50% or greater reduction in the mean cumulative score overseveral days following the onset of disease symptoms in comparison tothe control group.

Furthermore, effective treatment of MS may be examined in severalalternative ways including, EDSS (extended disability status scale),appearance of exacerbations, or MRI. Satisfying any of the followingcriteria evidences effective treatment.

The EDSS is a means to grade clinical impairment due to MS (Kurtzke(1983) Neurology 33:1444). Eight functional systems are evaluated forthe type and severity of neurologic impairment. Briefly, prior totreatment, impairment in the following systems is evaluated: pyramidal,cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral, andother. Follow-ups are conducted at defined intervals. The scale rangesfrom 0 (normal) to 10 (death due to MS). A decrease of one full stepdefines an effective treatment in the context of the present invention(Kurtzke (1994) Ann. Neurol. 36:573-79).

Exacerbations are defined as the appearance of a new symptom that isattributable to MS and accompanied by an appropriate new neurologicabnormality (IFN-β MS Study Group, supra). In addition, the exacerbationmust last at least 24 hours and be preceded by stability or improvementfor at least 30 days. Standard neurological examinations result in theexacerbations being classified as either mild, moderate, or severeaccording to changes in a Neurological Rating Scale (Sipe et al. (1984)Neurology 34:1368). An annual exacerbation rate and proportion ofexacerbation-free patients are determined. Therapy is deemed to beeffective if there is a statistically significant difference in the rateor proportion of exacerbation-free patients between the treated groupand the placebo group for either of these measurements. In addition,time to first exacerbation and exacerbation duration and severity mayalso be measured. A measure of effectiveness as therapy in this regardis a statistically significant difference in the time to firstexacerbation or duration and severity in the treated group compared tocontrol group.

MRI can be used to measure active lesions using gadolinium-DTPA-enhancedimaging (McDonald et al. (1994) Ann. Neurol. 36:14) or the location andextent of lesions using T₂-weighted techniques. Briefly, baseline MRIsare obtained. The same imaging plane and patient position are used foreach subsequent study. Areas of lesions are outlined and summed slice byslice for total lesion area. Three analyses may be done: evidence of newlesions, rate of appearance of active lesions, and percentage change inlesion area (Paty et al. (1993) Neurology 43:665). Improvement due totherapy is established when there is a statistically significantimprovement in an individual patient compared to baseline or in atreated group versus a placebo group.

It is further recognized that additional compounds can be administeredwith the cytokine to produce a therapeutic effect. For instance, IGF-1has been implicated in preventing the depletion of matureoligodendrocytes and promoting recovery from demyelination in MS andother demyelinating disorders. See, for example, Mason et al. (2000) J.Neuroscience 20:5703-5708, herein incorporated by reference. Hence,IFN-β can be administered in conjunction with IGF-1 for the treatment ofMS. The compounds can be administered by the methods of the invention.Alternatively, one of the compounds can be administered by any methodknown in the art including, for example, subcutaneous and intramuscularroutes.

The IGF-1 used according to the methods of the present invention can bein its substantially purified, native, recombinantly produced, orchemically synthesized forms. For example, IGF-1 can be isolateddirectly from blood, such as from serum or plasma, by known methods.See, for example, Phillips (1980) New Eng. J. Med. 302:371-380; Svobodaet al. (1980) Biochemistry 19:790-797; Cornell and Boughdady (1982)Prep. Biochem. 12:57; Cornell and Boughdady (1984) Prep. Biochem.14:123; European Patent No. EP 123,228; and U.S. Pat. No. 4,769,361.IGF-1 may also be recombinantly produced in the yeast strain Pichiapastoris and purified essentially as described in U.S. Pat. Nos.5,324,639, 5,324,660, and 5,650,496 and International Publication No. WO96/40776; all of which are herein incorporated by reference.

Alternatively, IGF-1 can be synthesized chemically, by any of severaltechniques that are known to those skilled in the peptide art. See, forexample, Li et al. (1983) Proc. Natl. Acad. Sci USA 80:2216-2220,Stewart and Young (1984) Solid Phase Peptide Synthesis (Pierce ChemicalCompany, Rockford, Ill.), and Barany and Merrifield (1980) The Peptides:Analysis, Synthesis, Biology, ed. Gross and Meienhofer, Vol. 2 (AcademicPress, New York, 1980), pp. 3-254, for solid phase peptide synthesistechniques; and Bodansky (1984) Principles of Peptide Synthesis(Springer-Verlag, Berlin); and Gross and Meienhofer, eds. (1980) ThePeptides: Analysis, Synthesis, Biology, Vol. 1 (Academic Press, NewYork), for classical solution synthesis. IGF-1 can also be chemicallyprepared by the method of simultaneous multiple peptide synthesis. See,for example, Houghten (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135;and U.S. Pat. No. 4,631,211. These references are herein incorporated byreference. Furthermore, methods to prepare a highly concentrated, lowsalt-containing, biologically active form of IGF-1 or variant thereofare provided in WO 99/24062, entitled Novel IGF-1 Compositions and ItsUse.

Methods for making IGF-1 fragments, analogues, and derivatives areavailable in the art. See generally U.S. Pat. Nos. 4,738,921, 5,158,875,and 5,077,276; International Publication Nos. WO 85/00831, WO 92/04363,WO 87/01038, and WO 89/05822; and European Patent Nos. EP 135094, EP123228, and EP 128733; herein incorporated by reference.

In addition, several IGF-1 variants are known in the art and includethose described in, for example, Proc. Natl. Acad. Sci. USA 83(1986):4904-4907; Biochem. Biophys. Res. Commun. 149 (1987):398-404; J.Biol. Chem. 263 (1988):6233-6239; Biochem. Biophys. Res. Commun. 165(1989):766-771; Forsbert et al. (1990) Biochem. J. 271:357-363; U.S.Pat. Nos. 4,876,242 and 5,077,276; and International Publication Nos. WO87/01038 and WO 89/05822. Representative variants include one with adeletion of Glu-3 of the mature molecule, a variant with up to 5 aminoacids truncated from the N-terminus, a variant with a truncation of thefirst 3 N-terminal amino acids (referred to as des(1-3)-IGF-1,des-IGF-1, tIGF-1, or brain IGF), and a variant including the first 17amino acids of the B chain of human insulin in place of the first 16amino acids of human IGF-1.

Meningitis refers to an inflammatory process of the leptomeninges andCSF within the subarachnoid space. Meningoencephalitis applies toinflammation of the meninges and brain parenchyma Meningitis is usuallycaused by an infection, but chemical meningitis may also occur inresponse to a non-bacterial irritant introduced into the subarachonoidspace. Infiltration of the subarachnoid space by carcinoma is referredto as meningeal carcinomatosis and by lymphoma as lymphomapyogenic(usually bacterial), aseptic (usually viral), and chronic (most anyinfectious agent).

It has been suggested that the central nervous system damage that occursin viral and bacterial meningitis may be more related to invasion of thesurface of the brain by the host's own lymphocytes in response to themeningitis pathogen, rather than to the pathogen itself or any toxinproduced by the pathogen (Lewis (1979) The Medusa and The Snail, PenguinBooks). In fact, many patients fall victim to the disease despite theprompt sterilization of the cerebrospinal fluid using the currentaggressive treatments, such as the third generation cephalosporins. Thisunexpected outcome may result from harmful interactions between hostcells/tissues and bacterial components released by treatment with lyticantibiotics (Scand et al. (1991) J. Infect., Dis. Supp. 74:173-179). Theburst of peptidoglycan, capsular polysaccharide, and lipopolysaccharideliberated from the bacteria induce the production of a number ofmediators including TNF in the central nervous system leading tomeningeal and perivascular inflammation in the subarachnoid space.Disruption of the blood-brain barrier ensues, leading to cerebral edema,ischemia, and a dramatic increase in intracranial pressure. Those thatsurvive the acute phase of the disease are often left with multipleneurological sequelae. Previous results from trials utilizingsteroid-based anti-inflammatories either prior to or concomitant withantibiotic administration suggest that such an approach may have value.See, for example, Mustafa et al. (1990) Amer. J. Diseases of Children144:883-887. Hence, administration of a cytokine, particularlyinterferon-β, using the methods of the present invention could beeffective in preventing damage by activated lymphocytes. The methods ofthe invention could be used in conjunction with the existing treatmentsfor meningitis to help prevent brain damage. Such treatments aredescribed in Harrison's Principles of Internal Medicine (McGraw Hill,1994), pp. 2296-2309, herein incorporated by reference.

An “effective amount” of a cytokine to treat meningitis using theadministration method of the present invention will be sufficient toreduce or lessen the clinical symptoms of meningitis. In preferredembodiments, the cytokine is administered in conjunction with anantibiotic regiment. As such, an effective amount of the cytokineaugments the activity of the antibiotics and leads to enhanced survivaland/or improved clinical status of the animals in comparison to animalstreated with antibiotics alone. Such clinical manifestations mayinclude, for example, 1) a more rapid normalization of the CNSinflammatory indices compared to a control; 2) a more rapiddisappearance in fever as compared to a control; 3) a reduction in theoverall neurologic sequelae; and/or, 4) an improved mortality ascompared to a control. More extensive details regarding the clinicalmanifestations of meningitis that can be improved upon theadministration of an effective concentration of a cytokine can be foundin Harrison's Principles of Internal Medicine (McGraw Hill, 1994), pp.2296-2309, herein incorporated by reference.

Primary Sjogren's Syndrome, also known as Dry Eye Syndrome, ischaracterized by decreased secretion of the lacrimal glands that makethe aqueous layer of the tear film that lubricates the eyes. Manypatients afflicted with Sjogren's Syndrome also experience dry mouth dueto decreased secretion of the salivary glands. This is an autoimmunedisease characterized by chronic inflammation and infiltration of thelacrimal and salivary glands by lymphocytes. Activated T cells of theCD4⁺ type that infiltrate the lacrimal gland mediate tissue destruction(Tabbara et al. (1999) Eur. J. Ophthalomol. 9:1-7). Recently,nHu-IFN-alpha administered by the oral mucosa route has been shown tostimulate output (Ship et al. (1999) J. Interferon Cytokine Res.19:480-488).

Hence, the present invention provides a method of administeringcytokines, particularly, IFN-α and IFN-β, such that the compoundsdirectly enter the nasal lymphatic system. The interferon will then bedistributed to the lymphatics of the head and neck region altering thefunction of the lymphocytes that affect the lacrimal and salivaryglands. It is further recognized that delivery of the cytokine via thetrigeminal or the olfactory nerve can result in the direct delivery ofthe cytokine to the lacrimal gland. This direct delivery of theinterferon to the lymphatics of the head and neck region or directly tothe lacrimal gland will reduce lymphocyte infiltration of the lacrimaland salivary glands and treat Sjogren's Syndrome.

An “effective amount” of a cytokine to treat Sjogren's Syndrome usingthe administration method of the present invention will be sufficient toreduce or lessen the clinical symptoms of Sjogren's Syndrome. As such,an effective amount of the cytokine leads to an improved clinical statusof a patient suffering from Sjogren's Syndrome in comparison to anuntreated patient. For instance, an improved clinical status of the oralsymptoms of Sjogren's Syndrome includes, for example, an overallincrease in mouth wetness, an improvement in the ability to swallow dryfood, an improvement in the ability to speak continuously, etc. Further,an effective concentration encompasses any improvement in the ocularmanifestations of Sjogren's Syndrome including, for example, increase inthe wetness of eyes (i.e., a lessening of the sandy or gritty feelingunder the eyelids), an increase in tearing, and a decrease in burningsensations, redness, itching, and eye fatigue. Improvements alsoencompass an improvement in lacrimal function (i.e., a reduction inlymphocyte infiltration into the lacrimal gland). A more extensivedescription of the clinical manifestation of Sjogren's Syndrome can befound in Harrison's Principles of Internal Medicine (McGraw Hill, 1994),pp. 1662-1664, herein incorporated by reference.

Treatment of Viral Infections

In another embodiment, the present method can be employed to delivercytokines and/or antiviral agents to the lymphatic system, CNS, brain,and/or spinal cord for the treatment, diagnosis or prevention ofdisorders or disease resulting from viral infection.

As used herein “treating or preventing viral infection” means to inhibitvirus transmission, or to prevent the virus from establishing itself inits host CNS, brain or spinal cord, or to ameliorate or alleviate thesymptoms of the disease caused by viral infection. The treatment isconsidered therapeutic if there is a reduction in viral load in the CNS,brain, or spinal cord, decrease in mortality, and/or morbidity. Ofparticular interest is the administration of a cytokine (particularlyIFN-α or IFN-β) by the methods of the invention for the treatment orprevention of viral hepatitis.

Viral hepatitis refers to an infection of the liver caused by a group ofviruses having a particular affinity for the liver and include hepatitisA virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, andhepatitis E virus. Of particular interest is the use of the presentinvention for the treatment of hepatitis C.

Acute infection with hepatitis C virus results in persistent viralreplication and progression to chronic hepatitis in approximately 90% ofcases. While chronic hepatitis C infection is commonly treated withIFN-β and IFN-α, less than 50% of the patients have sustained remissionfollowing treatment (i.e., the eradication of hepatitis C virus). See,for example, Barbaro et al. (1999) Scand. J. Gastroenterol. 9:928-933;Oketani et al. (1999) J. Clin. Gastroenterol. 28:49-51; and, Kakizaki etal. (1999) J. Viral Hepatitis 6:315-319; all of which are hereinincorporated by reference. Similarly, IFN therapy has also beendemonstrated to be an effective treatment for chronic hepatitis B,however only 25-40% of the patients profit from a long-term beneficialresponse to the current interferon therapies. Combination therapies forviral hepatitis have also been developed, which combine IFN-therapy withantiviral agents such as ribavirin. These IFN/antiviral therapies areusually given systemically (i.e., intravenously), and hence, thetherapeutic agents are not able to cross the blood-brain barrier. Thus,the hepatitis virus can harbor in the central nervous system where thetherapeutic agents cannot penetrate. Re-infection and relapse to viralhepatitis symptoms subsequent to treatment frequently occurs. Inaddition, viral hepatitis infection of the CNS can have seriousneurologic consequences. See, for example, Bolay et al. (1996) Clin.Neurol. Neurosurg. 98:305-308, herein incorporated by reference.Therefore, new methods of treatment are necessary in the treatment ofviral hepatitis. The methods of the present invention can be used toadminister a cytokine and/or an antiviral agent or any combinationthereof, to the lymphatic system, CNS, brain and/or spinal cord for thetreatment or prevention of viral hepatitis. The methods of the inventioncan be used in conjunction with the existing treatments for viralhepatitis to aid in reducing the clinical symptoms of hepatitis.

As used herein, an “effective amount” of a cytokine or an antiviralagent for the treatment of viral hepatitis using the administrationmethod of the present invention will be sufficient to reduce or lessenthe clinical symptoms of hepatitis. As such, an effective amount of thecytokine or antiviral agent administered by the methods of the presentinvention will augment the activity of the systemically administeredantiviral/immunomodulatory compounds used in the art for the treatmentof viral hepatitis. As such, the methods of the invention enhancesurvival and/or improve clinical status of the treated animals incomparison to animals treated with systemic administration methodsalone. Improvement in clinical status includes, for example, theprevention of the progression of acute viral hepatitis to chronicity,the reduction of the viral load in chronic hepatitis, and/or theprevention or reduction in the frequency of re-infection and relapse ofviral hepatitis symptoms, and/or prevent or reduce the neurologic damageresulting from the viral infection.

Antiviral agents and cytokines of particular interest include, forexample, ribavirin, thymosins, and cytokines, such as, IFN-β, IFN-α, andIFN-γ. See, for example, Musch et al. (1998) Hepato-Gastroenterology45:2282-2294; Barbaro et al. (1999) Scand. J. Gastroenterol.9(34):928-933; Oketani et al. (1999) J. Clin. Gastroenterol. 28:49-51;Kakizaki et al. (1999) J. Viral Hepatitis 6:315-319; U.S. Pat. No.6,030,785; U.S. Pat. No. 5,676,942; and U.S. Pat. No. 6,001,799; all ofwhich are herein incorporated by reference.

The course of the viral hepatitis and its response to the treatmentsadministered by the methods of the present invention may be followed byclinical examination and laboratory findings that are commonly performedin the art. For instance, elevated serum alanine aminotransferase (ALT)and aspartate aminotransferase (AST) are known to occur in uncontrolledhepatitis C. A complete response to treatment is generally defined asthe normalization of these serum enzymes, particularly ALT (Davis et al.(1989) New England J. Med. 321:1501-6). Alternatively, hepatitis C virusreplication in subjects in response to the antiviral/immunomodulatorytreatment of the present invention can be followed by measuringhepatitis C virus RNA in serum samples by, for example, a nestedpolymerase chain reaction assay that uses two sets of primers derivedfrom the NS3 and NS4 non-structural gene regions of the HCV genome(Farci et al. (1991) New England J. Med. 325:98-104; Ulrich et al.(1990) J. Clin. Invest. 86:1609-14).

In another embodiment, the methods of the present invention can be usedto treat or prevent herpes simplex viral infection. Herpes simplexviruses (HSV-1 and HSV-2) produce a variety of infections involvingmucocutaneous surfaces, the central nervous system, and occasionallyvisceral organs. For instance, acute viral replication at a peripheralsite such as the cornea is followed by viral entry into neuronaltermini. Corneal infection is followed by intra-axonal transport, whichmoves the virus to the trigeminal ganglia, where further replication mayoccur before clearance of infectious virus and the establishment oflatency. Failure to clear the virus may result in central nervous systeminfection, encephalitis, and death. Latency may periodically break downin response to certain stimuli, leading to viral reactivation andshedding. The present invention provides a method of administering acytokine (via, for example, the trigeminal or olfactory nerve) to thetrigeminal ganglia and/or the CNS, thereby allowing for the treatmentand/or prevention of herpes simplex viral infection.

The immune response to acute herpes simplex virus infection involvesboth innate and acquired immunity. Key mediators of innate resistance toviral infection include cytokines, particularly interferons such asIFN-α, IFN-β, and IFN-γ. For instance, IFN-α has been shown to inhibitthe onset of immediate-early herpes simplex virus gene expression(Oberman et al. (1988) J. Gen. Virol. 69:1167-1177). Furthermore, inmice IFN-α and IFN-β are potent inhibitors of replication in the cornea.Specifically, studies have shown that following corneal inoculation inmice, herpes simplex viral titer in both the eyes and trigeminal gangliawas enhanced by up to 1000 fold in mice mutant for IFN-α or IFN-βcompared to wild-type control mice (Leib et al. (1999) J. Exp. Med.189:663-672, herein incorporated by reference). The same study furtherdemonstrated that IFNs significantly reduce productive viral infectionand reduce the spread of virus from intact corneas. Related studies havealso been preformed by Minagawa et al. (1997) Antiviral Res. 36:99-105.

In addition, IFN-α and IFN-β activate host defenses such as naturalkiller cells, which have themselves been shown to be important incontrolling herpes simplex virus infection and pathology (Bouley et al(1996) Clin. Immunol. Immunopathol. 80:23-30). IFN-α and IFN-β have alsobeen suggested to be important for limiting progress of infection fromperipheral tissues to the nervous system (Halford et al. (1997) Virology236:328-337). Furthermore, IFN-γ appears to play an important role inthe clearance of herpes simplex virus from the cornea and in resistanceto encephalitis, possibly by inhibiting apoptosis of neurons (Bouley etal. (1995) J. Immunol. 155:3964-3971, Geiger et al. (1997) Virology238:189-197, and Imanishi et al. (2000) J. Biochem. 127:525-530). Hence,interferons, particularly IFN-α, IFN-β, and IFN-γ, play a major role inlimiting herpes simplex viral replication in the cornea, trigiminalganglia, and in the nervous system.

An “effective amount” of a cytokine for the treatment of herpes simplexvirus using the administration method of the present invention will besufficient to reduce or lessen the clinical symptoms of herpes simplexvirus. As such, an effective amount of the cytokine administered by themethods of the present invention will attenuate the activity of thevirus and thereby enhance survival and/or improve clinical status of thetreated animal in comparison to the untreated control. Improvement inclinical status includes, for example, the prevention or reduction ofencephalitis and/or apoptosis in the central nervous system (i.e,increase in neuroprotection), a decrease in the severity of infection(i.e., enhancing viral clearance from the cornea, the trigeminalganglia, and the CNS), a decrease in viral spread, an increase in themaintenance of latency, and/or a decrease in the frequency of herpessimplex recurrences. More extensive details regarding the clinicalmanifestations of herpes simplex that can be improved upon theadministration of an effective concentration of a cytokine can be foundin Harrison's Principles of Internal Medicine (McGraw Hill, 1994), pp.782-787, herein incorporated by reference.

In another embodiment, the methods of the invention can be used for thetreatment of human immunodeficiency virus (HIV). HIV is an infectiousdisease of the immune system characterized by a progressivedeterioration of the immune system in most infected subjects. Duringdisease progression, key cells associated with the immune system becomeinfected with HIV, including, e.g., CD4⁺ T cells, macrophages/monocytes,and glial cells. Prolonged HIV infection frequently culminates in thedevelopment of AIDS. In the late stages of this disease, the immunesystem is severely compromised due to loss or dysfunction of CD4⁺ Tcells (Shearer et al. (1991) AIDS 5:245-253). The nervous system is alsoa major target of HIV infection. The virus is carried to the brain byinfected monocytes and the neurologic manifestations of HIV infectionare thought to arise from viral products and soluble factors produced bythe infected macrophages/microglia. Thus, the HIV virus can harbor inthe central nervous system where the therapeutic agents cannotpenetrate. Re-infection and relapse to HIV symptoms subsequent totreatment frequently occurs. Accordingly, the present invention providesa method of administering a cytokine, particularly an interferon such asIFN-α, IFN-β, and IFN-γ, to the CNS or the lymphatic system for thetreatment or prevention of HIV infection.

Interferons are known to exert pleiotropic antiretroviral activities andaffect many different stages of the HIV infectious cycle. For instance,IFN-β influences uptake of HIV particles (Vieillard et al. (1994) Proc.Natl. Acad. Sci. USA 91:2689-2693); reverse transcription of viralgenomic RNA into proviral DNA (Baca-Regen et al. (1994) J. Virol.68:7559-7565: Kornbluth et al. (1990) Clin. Immunol. Immunopathol.54:200-219 and Shirazi et al. (1993) Virology 193:303-312); viralprotein synthesis (Coccia et al. (1994) J. Biol. Chem. 269:23087-23094);and packaging and release of viral particles (Poli et al. (1989) Science244:575-577). In addition, virions released from IFN-β treated cells areup to 1,000-fold less infectious than equal numbers of virions releasedfrom untreated cells (Hansen et al. (1992) J. Virol. 66:7543-7548).Furthermore, recent studies have shown that genetically engineered humanCD4⁺ T cells producing constitutively low amounts of IFN-β can eradicateHIV in vivo using a mouse animal model that supports persistent,replicative HIV infection. These results indicated that a therapeuticstrategy based upon IFN-β transduction of CD4⁺ T cells may be successfulin controlling a preexisting HIV infection and allowing immunerestoration. See, for example, Vieillard et al. (1999) J. Virol.73:10281-10288, herein incorporated by reference. IFN-γ has also beenshown to modulate the susceptibility of macrophages to HIV (Zaitseva etal. (2000) Blood 96:3109-3117).

It is recognized that administration of the cytokine via the methods ofthe present invention for the treatment of HIV can be used incombination with any other HIV treatment or therapy known in the art.Therapies used in the treatment of HIV infection include, for example,anti-retroviral drugs, such as reverse transcriptase inhibitors, viralprotease inhibitors, and viral entry inhibitors (Caliendo et al. (1994)Clin. Infect. Dis. 18:516-524). More recently, treatment withcombinations of these agents, known as highly active antiretroviraltherapy (HAART), has been used to effectively suppress replication ofHIV (Gulick et al. (1997) N. Engl. J. Med. 337:734-9 and Hammer et al.(1997) N. Engl. J. Med. 337:725-733).

An “effective amount” of a cytokine for the treatment of HIV using theadministration method of the present invention will be sufficient toreduce or lessen the clinical symptoms of HIV. As such, an effectiveamount of the cytokine administered by the methods of the presentinvention will attenuate the activity of the virus (i.e., have a directantiviral effect) and/or improve the HIV-induced immunologicaldysfuntions (i.e., enhance the ability of an HIV-infected patient toeffectively mount a cellular immune defense against actively replicatingHIV). Regardless of the mechanism of action, an effective amount of acytokine will enhance survival and/or improve clinical status of thetreated animals in comparison to the untreated control. Improvement inclinical status includes, for example, a reduction in preexisting HIVinfection and/or the rate of disease progression; enhanced CD4⁺ T-cellsurvival; suppression of cytokine dysregulation caused by HIV (i.e.,enhanced Th1-like cytokine expression); inhibition of viral replication;and improvement in the proliferative expansion of antigen-selectedlymphocytes, more particularly the HIV antigen-specific CD8⁺ subset of Tcells, in response to an increase in viral load. Assays to measure thesevarious improvements are known in the art. See, for example, Vieillardet al. (1999) J. Virol. 73:10281-10288, Vieillard et al. (1997) Proc.Natl. Acad. Sci. USA 94:11595-11600; U.S. Pat. No. 5,911,990 and U.S.Pat. No. 5,681,831; all of which are herein incorporated by reference.More extensive details regarding the clinical manifestations of HIV thatcan be improved upon the administration of an effective concentration ofa cytokine can be found in Harrison's Principles of Internal Medicine(McGraw Hill, 1994), pp. 1559-1617, herein incorporated by reference.

Treatment of Proliferative Disorders of the CNS

In another embodiment, the present method can be employed to delivercytokines to the lymphatic system, CNS, brain, and/or spinal cord forthe treatment, diagnosis or prevention of a proliferation disorder ordisease.

Cytokines have anti-proliferative activity. For instance, interferonshave been shown to have both a direct cytotoxic effect on tumor cellsand an indirect cytotoxic effect through the activation of naturalkiller cells, macrophages, or other immune cells. Specifically, studieshave suggested IFN-γ mediated anti-tumor activity results frommodulating the interplay of B and T cell components of the immunesystem, as well as the inhibition of tumor angiogenesis (Saleh et al.(2000) Gene Ther 7:1715-24). IFN-α has also been shown to significantlydecrease average tumor size and increase the average survival time ofthe treated mammal (Wang et al. (1999) J Neuropathol Exp. Neurol.58:847-58). Intratumoral injection of liposomes containing the humanIFN-β gene in nude mice inhibits tumor growth, with complete tumorregression occurring following multiple Intratumoral injections of thegene. Furthermore, IFN-β has been demonstrated to be an effectivetreatment of high grade astrocytomas (Natsume et al. (1999) Gene Ther.9:1626-33 and Fine et al. (1997) Clin. Cancer Res 3: 381-7). Theantiproliferative effect of IFN-β appears to occurs through an arrest inthe ordered progression through S phase or decreasing the entry intoG2/M phase of the cell cycle (Garrison et al. (1996) J Neurooncol30:213-23). Hence, interferons, particularly IFN-α, IFN-β, and IFN-γ,are effective agents for the treatment or prevention of a proliferationdisorder of the CNS, spinal cord, brain and lymphatic system.

By “proliferation disorder” is intended any disorder characterized bycellular division occurring in defiance of the normal tissue homeostasismechanism. The proliferation disorder can be either malignant or benignand result from either an increase in the rate of cell proliferation ora decrease in the rate of cell death. The proliferative disorder treatedby the methods of the invention may be at any stage of development(i.e., an early stage with minimal or microscopic tumor burdens or atadvanced stages of tumor development).

Proliferative disorders of the central nervous system, brain, or spinalcord include, for example, gliomas, neuronal tumors, poorlydifferentiated neoplasms, and meningiomas. Gliomas derived from glialcells include astrocytomas (i.e., fibrillary astrocytomas, glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthastrocytoma, andbrain stem glioma), oligodendrogliomas, and ependymomas andparaventricular mass lesions (i.e., myxopapillary ependymomas,subependymomas, choroid plexus papillomas). Neural tumors comprise CNStumors that contain mature-appearing neurons (ganglion cells) that mayconstitute the entire cell population of the lesion or, alternatively,the lesion is an admixture with a glial neoplasm. Poorly differentiatedneoplasms include, for example, medulloblastomas. Other proliferativedisorders of the CNS, brain or spinal cord include, primary brainlymphoma, meningiomas, and metastatic tumors.

It is recognized that administration of the cytokine via the methods ofthe present invention for the treatment of a proliferative disorder canbe used in combination with any other treatment or therapy known in theart for the treatment of proliferation disorders. Therapies used in thetreatment of proliferative disorders include, for example, any form ofradiation and chemotherapy treatments. See, for example, Hatano et al.(2000) Acta Neurochir 142:633-8, Burton et al. (1999) Curr Opin Oncol.11:157-61, and Brandes et al. (2000) Anticancer Res 20:1913-20; all ofwhich are herein incorporated by reference.

An “effective amount” of a cytokine for the treatment of a proliferativedisease or disorder using the administration method of the presentinvention will be sufficient to reduce or lessen the morphologicaland/or clinical symptoms of the proliferative disorder. As such, aneffective amount of the cytokine administered by the methods of thepresent invention will exert any physiological response that decreasesproliferation of tumor cells and thereby enhances survival and/orimproves clinical status of the treated animal in comparison to theuntreated control. Such physiological responses include, for example,activation of immune cells, inhibition of cell proliferation, inductionof cell differentiation, up-regulation of class I majorhistocompatiblity complex antigens, inhibition of angiogenesis, andestablishment of the T helper 1 (Th1)-type response. Improvement inclinical status includes, for example, an increase in the survival rateof the treated mammal (i.e., an increase in either the one or two yearsurvival rate) and a decrease in tumor size. Assays to measure thesevarious improvements are known in the art. See, for example, Hong et al.(2000) Clin. Cancer Res. 6:3354-60); Knupfer et al. (2000) Cytokine12:409-12; Natsume et al. (1999) Gene Ther 6:1626-33; and U.S. Pat. No.4,846,782, all of which are herein incorporated by reference. Moreextensive details regarding the clinical manifestations of proliferativedisorders of the CNS, brain, spinal cord, or lymphatic system that canbe improved upon the administration of an effective concentration of acytokine can be found in Harrison's Principles of Internal Medicine(McGraw Hill, 1994), herein incorporated by reference.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXPERIMENTAL Example 1 Intranasal Administration of IFN-β to the CNS

Introduction

Administering interferon-β (IFN-β) intranasally is an effective meansfor delivering this cytokine to the CNS of an animal.

Materials and Methods

Intranasal Delivery to the CNS:

Male Sprague-Dawley rats, 199 and 275 grams, were anesthetized withintraperitoneal pentobarbital (40 mg/kg). Drug delivery to the CNS wasassessed after intranasal administration of 51 picomoles and 57picomoles of ¹²⁵I-IFN-β in 20 mM Hepes, pH 7.5, to the light and heavyrat, respectively. Rats were placed on their backs and administered ˜100microliters ¹²⁵I-IFN-β to each naris over 10-22 minutes, alternatingdrops every 2-3 minutes between the left and right nares. During theintranasal administration of IFN-β, one side of the nose and the mouthwere held closed. This method of administering the cytokine allows forboth pressure and gravity to deliver the agent into the upper one thirdof the nasal cavity. Rats subsequently underwent perfusion-fixationwithin minutes following the completion of ¹²⁵I-IFN-β administration.Perfusion-fixation was performed with 50-100 ml physiologic salinefollowed by 500 ml of fixative containing 4% paraformaldehyde in 0.1 MSorenson's phosphate buffer, pH 7.4, prior to brain and spinal corddissection and ¹²⁵I measurement by gamma counting. Areas dissectedincluded the spinal cord, olfactory bulbs, frontal cortex, anteriorolfactory nucleus, hippocampal formation, choroid plexus, diencephalon,medulla, pons, and cerebellum.

Results

Rapid appearance of radiolabel was observed throughout the spinal cord,brain stem, and brain, with the concentrations ranging from about 3 pMto about 93 pM. Detailed results are shown below in Table 1. Theobservation of substantial concentrations of interferon-β in theolfactory and trigeminal nerves suggests that this cytokine istransported through or along these nerves. Tissues with biologicallysignificant levels of interferon-β include the olfactory bulbs, frontalcortex, caudate putamen, anterior olfactory nerve, hippocampalformation, choroid plexus, diencephalon, pons, medulla, ventral dura,trigeminal nerve, olfactory epithelium, circle of Willis, and uppercervical spinal cord. TABLE 1 Data for the intranasal (I.N.) delivery ofBetaseron to the CNS Concentration (pM) Concentration (pM) Tissue type(51 picomole dose) (57 picomole dose) Left olfactory bulb 89.5 51.4Right olfactory bulb 92.9 67.7 Frontal cortex 9.19 29.1 Caudate/putamen7.09 34.0 Anterior olfactory nerve 46.9 97.4 Hippocampal formation(left) 5.81 11.7 Hippocampal form (right) 11.1 21.0 Choroid plexus 79.033.2 Diencephalon 15.5 24.0 Midbrain 10.9 19.8 Pons 16.9 49.4 Medulla24.7 90.2 Cerebellum 10.2 30.4 Dura ventral 263.0 896 Trigeminal nerve36.7 362 Left olfactory epithelium 3697 Circle of Willis 189 UpperCervical Spinal Cord 24.3 455 Cervical spinal cord 6.88 Thoracic spinalcord 4.0 2.55 Lumbar spinal cord 2.08 3.5 Right olfactory epithelium22,540

Further quantitation studies for the intranasal delivery of[¹²⁵I]Betaseron were performed in Sprague-Dawley rats essentiallydescribed above. The results are summarized in Table 2. Scans of coronalbrain tissue sections showed prominent labeling of the olfactory bulb,caudate/putamen, septal nucleus, periventricular white matter, opticnerve, and superior colliculus (data not shown). These results are inagreement with the results provided in Table 1. The quantitative studiesperformed in six animals, following internasal administration of about 6nmol of Betaseron, demonstrated consistent delivery to a wide variety ofCNS structures. Highest concentrations of IFN-β were found in theolfactory bulbs (9 nM), anterior olfactory nucleus (3.3 nM), midbrain(1.9 nM), medulla (1.8 nM), pons (1.6 nM), and cerebellum (1.4 nM).Moderate concentrations were observed in the hippocampal formation (1.3nM), diencephalon (1.3 nM), frontal cortex (1.1 nM), cervical spinalcord (1.1 nM), and caudate/putamen (0.83 nM).

The very high concentrations of [¹²⁵I]Betaseron observed in thetrigeminal nerve (14 nM) and ventral dura mater (19 nM) strongly suggestthat delivery to the CNS involves movement not only along the olfactoryneural pathway but also along the trigeminal nerve pathway. Trigeminaldelivery should result in high levels in both the olfactory areas andmidbrain and brain stem regions. Delivery to the spinal cord probablyoccurs via the trigeminal pathway. Consistent with trigeminal delivery,[¹²⁵I]Betaseron reaches the spinal cord within 25 minutes, and exhibitsdecreasing concentration as you move down the spinal cord.

These results indicate the direct transport of IFN-β along one or moreneural pathways into the CNS, brain, and spinal cord. TABLE 2Concentration (nM) IFN-β (Betaseron) in Different Rat Tissues FollowingI.N. Administration of ¹²⁵I-IFN-β + IFN-β Tissue IF11 IF12 IF13 IF14IF15 IF16 Mean SE Blood Sample 1 1.12 1.53 0.92 0.74 1.70 0.85 1.1 0.2Blood Sample 2 2.77 3.26 1.44 2.11 3.13 2.74 2.6 0.3 Blood Sample 3 3.726.62 2.81 3.87 5.02 4.22 4.4 0.5 Blood Sample 4 5.37 6.35 7.38 5.4 7.325.50 6.2 0.4 Blood Sample 5 7.29 6.69 8.15 7.95 7.5 0.3 Left OlfactoryBulb 9.02 6.11 3.01 5.93 18.29 18.46 10 3 Right Olfactory Bulb 5.6 6.993.39 4.84 15.26 12.48 8.1 1.9 Frontal Cortex 1.12 1.24 1.09 0.44 1.721.19 1.1 0.2 Caudate/Putamen 0.68 0.91 0.83 0.36 1.08 1.1 10.83 0.11Ant. Olf. Nucleus 2.11 2.55 1.96 1.09 6.82 5.50 3.3 0.9 L. HippocampalForm. 0.84 1.63 1.24 0.37 2.23 1.71 1.3 0.3 R. Hippocampal Form. 0.851.77 1.24 0.40 1.84 1.91 1.3 0.3 Diencephalon 0.86 1.52 1.39 0.44 2.051.72 1.3 0.2 Midbrain 0.80 1.69 1.53 0.44 5.07 1.91 1.9 0.7 Pons 0.761.91 1.76 0.38 2.71 2.04 1.6 0.4 Medulla 0.63 2.41 2.90 0.42 2.29 2.081.8 0.4 Cerebellum 0.89 1.72 1.56 0.36 2.19 1.84 1.4 0.3 Ventral Dura2.47 46.16 10.89 7.13 21.35 23.52 19 6 Trigeminal Nerve 7.94 12.14 19.894.57 24.44 17.63 14 3 Spinal Dura 0.59 0.13 0.29 0.34 0.13 CervicalSpinal Cord 0.33 0.88 3.12 0.38 0.98 1.00 1.1 0.4 Thoracic Spinal Cord0.14 0.11 0.39 0.29 0.33 0.15 0.24 0.05 Lumbar Spinal Cord 0.13 0.120.27 0.22 0.32 0.10 0.19 0.04 Deltoid Muscle 0.62 0.58 0.50 1.10 0.670.22 0.62 0.12 Liver 0.58 0.78 1.01 1.38 0.54 0.31 0.77 0.16 Kidney 0.670.73 2.08 5.26 0.56 1.81 1.9 0.7 Lung 1.87 0.56 2.18 0.72 0.85 0.99 1.20.3 Esophagus 1.10 1.50 68.2 5234.83 1.44 22.40 888 869 Trachea 1.483.11 83.46 4.67 1.45 5.91 17 13 L. Olfact. Epithelium 1175.9 75.64 14.081431.14 454.41 227.29 563 244 R. Olfact. Epithelium 2083.1 411.32 45.661113.87 191.13 2765.47 1102 453IF11-16 represent individual ratsAverage weight (g.) across rats: 243 g. (range = 203 g − 268 g)Average concentration administered: 6.0 n moles (range = 4.8 nmol − 6.9nmol)Average radioactivity (uCi): 39 uCi (range = 32 uCi − 52 uCi)

Example 2 Intranasal Administration of IFN-β Retains PharmacologicalActivity in the CNS

Assays were performed to determine if IFN-β, delivered intranasally,retained pharmacological activity in the CNS. IFN-β activates signaltransduction pathways via a cell surface IFN receptor. The IFN receptoris part of a prototypical JAK-STAT signaling complex. It has twotransmembrane chains that associate with intracellular signalingproteins including TYK2, JAK1, and two latent transcription factorstermed “signal transducers and activators of transcription” (STATs).Binding of IFN-β to the receptor brings the two Janus kinases (TYK2 andJAK1) near each other, and they become activated by phosphorylation. Thekinases then activate the cytoplasmic tails of the IFN receptors byphosphorylating tyrosine residues. These phosphotyrosines providedocking sites for the STATs, bringing them into appropriate positionsfor phosphorylation by the nearby Janus kinases. Upon phosphorylationSTATs translocate to the nucleus, bind specific DNA elements and directtranscription. Hence, the pharmacological activity of IFN-β followingintranasal delivery can be effectively assayed by monitoring thephosphorylation states of TYK2 and STAT1 throughout the brain cortex.

Methods:

Control/Drug Treatment:

Harlan Sprague-Dawley rats were anesthetized with pentabarbitol (50mg/kg). 80 μl of either water or IFN-β was intranasally administered in5 doses over a 20 minute time period. Specifically, 8 μl wasadministered in 5 doses at 2 minute intervals for each nostril.Recombinant rat interferon-# (rrIFN-β (35 picomoles) was intranasallyadministered to rat IF35 (drug-treated) and H₂O (vehicle used to diluterrIFN-β) was administered to rat IF33 (control-treated). Afteradministration the animal was perfused with 100 ml of saline and fixedwith 200 ml of 10% formalin. The brain was then removed and sliced in abrain matrix into 2 mm sections. The slices were collected in cassettesand paraffin embedded. Tissue was sliced to 4 μm and placed onmicroscope slides.

Immunohistological Staining:

The antibodies to the phosphorylated forms of proteins TYK2 and STAT1were purchased from Cell Signaling Technology (product numbers 9321L and9171S, respectively).

The method of immunohistological staining was as follows. Tissuesections were deparaffinized and hydrated by placing the slides in thefollowing solutions for the indicated times: Xylene for 10 min; 100%EtOH for 5 min; 95% EtOH for 5 min; 70% EtOH for 5 min; and, 50% EtOHfor 5 min. The slides were removed from Coplin jars and washed in H₂Ofor 2 min on a rocking platform. The antigen (TYK2 and/or STAT1) wasunmasked by incubating slides in citrate buffer (pH 6.0) and heating ina vegetable steamer for 45 min. The slides were removed and washed incold running tap H₂O for 10 min. Slices were incubated in 3% H₂O for 10min at room temperature (RT) in a humid chamber and subsequently washedin H₂O for 5 min. Next, slides were washed in a tris buffered salinesolution (50 mM tris, 150 mm NaCl) with 0.2% Triton X-100 (TBST) forthree 5 min washes. Following the wash, the slides were blocked with 2%goat serum in TBST (GSTBST) for 1 hr at RT. Following three 5 min washesin TBST, the slides were incubated with primary antibody (rabbitanti-TYK2 polycolonal antibody; diluted in GSTBST 1:250) in a humidchamber at RT for 30 min and incubated overnight at 4° C. The next day,the slides were wash in TBST for three 5 min washes and incubated withgoat anti-rabbit secondary antibody. The secondary antibody was diluted1:400 in 10 mM phosphate buffered solution (PBS; 137 mM sodium chloride,2.7 mM potassium chloride) at RT for 1 hr. For the last 15 min of thisincubation, the ABC reagent was made (5 ml PBS, 2 drops of reagent A,mix, 2 drops of reagent B, mix; Vector Technology product # PK-6101) andallowed to stand at RT. Slides underwent an additional three 5 minutewashes in TBST, followed by incubation with ABC reagent at RT for 1 hrin a humid chamber. An additional three 5 minute washes in TBSTfollowed. Approximately 100-150 μl, enough to cover the tissue, ofdiaminobenzidine tetrahydrochloride (DAB) was added and allowed toincubate at RT for 10 min. The reaction was stopped by a 2 minute washwith H₂0. Slides were subsequently washed in H₂0 until the solution wasclear. Slides were dehydrated in the following solutions for theindicated times: 50% EtOH for 2 min; 70% EtOH for 2 min; 95% EtOH for 2min; 100% EtOH for 2 min; 50/50 Xylene/ROH for 2 min; and Xylene for 5min. Excess xylene was removed and slides were mounted by adding 2-3drops of Vectamount and covered with coverslip. The Vectamount wasallowed to dry before viewing.

Results:

Induction of the IFN-α/β pathway is characterized by the phosphorylationof TYK2 and STAT1. Therefore, antibodies specific to the phosphorylatedforms of TYK2 and STAT1 were used to measure the level of the activatedfrom of these proteins prior to and following intranasal delivery ofIFN-β. Quantitation revealed that the levels of phosphorylated TYK2increased throughout the brain cortex following intranasal delivery of35 pmol of recombinant rat IFN-β (data not shown). These resultsdemonstrate that IFN-β retains pharmacological activity in the CNSfollowing the intranasal delivery methods of the present invention.

Example 3 Intranasal Administration of IFN-β to the Lymphatic System

Intranasal delivery of [¹²⁵I] Betaseron was performed in Sprague-DawleyRats as essentially described in Example 1. 3.9-7.9 nmol Betaseron wasadministered in a 44-96 μl volume over the course of 20-29 minutes.Animals were perfused at 30 minutes. Data obtained from eight individualanimals is provided in Table 3. Experimental means from this set ofexperiments are provided in Table 4. These quantitative studiesdemonstrated delivery of [¹²⁵I] Betaseron to the superficial cervicalnodes and to the deep cervical nodes of the lymphatic system. Onaverage, 6.1 nM Betaseron was found in the superficial cervical nodes,and 31.5 nM was found in the deep cervical nodes following theadministration methods of the invention. These results are summarized inTable 5. TABLE 3 Betaseron Concentration (nM) Following I.N.Administration of ¹²⁵I-IFNβ + rhIFNβ Experiment 1F34 1F36 1F37 1F38MicroCi 31 47 61 48 Nmol 3.9 6.9 7.9 6.6 Blood Sample #1 0.53 0.58 1.11.6 Blood Sample #2 1.6 4.3 2.8 3.9 Blood Sample #3 2.4 3.1 4.6 6.2Blood Sample #4 3.6 4.7 5.1 8.3 Blood Sample #5 4.1 7.0 7.0 10 BloodSample #6 8.2 8.5 10 Left Olfactory Epithelium 65 862 388 643 RightOlfactory Epithelium 62 1103 1447 1876 Left Olfactory Bulb 1.6 3.5 5.6Right Olfactory bulb 1.3 8.1 7.1 Anterior Olfactory Nucleus 0.96 1.3 2.5Frontal Cortex 0.28 0.84 0.97 Caudate/Putamen 0.09 0.57 1.7 L+RHippocampus 0.38 0.62 0.82 Left Hippocampus Right HippocampusDiencephalon 0.65 0.74 0.95 Midbrain 0.48 0.61 0.88 Pons 0.45 0.75 0.91Medulla 0.36 0.76 0.95 Cerebellum 0.34 0.54 0.69 Ventral Brain Dura 6.19.7 12 14 Optic Nerve + Chiasm 1.2 6.3 4.4 25 Trigeminal Nerve 5.8 128.5 20 Spinal Dura 0.09 0.16 0.67 1.1 Upper Cervical Cord 0.43 2.3 0.921.1 Cervical Spinal Cord 0.17 0.21 0.47 1.3 Thoracic Spinal Cord 0.130.20 0.35 0.58 Lumbar Spinal Cord 0.17 0.29 0.39 0.49 SuperficialCervical Nodes 8.1 6.3 4.0 6.1 L. Superficial Cervical Node 3.9 R.Superficial Cervical Node 4.2 Deep Cervical Nodes 9.7 16 68 Left DeepCervical Node Right Deep Cervical Node Common Carotids 14 27 38 22Thyroid 250 462 830 725 Esophagus 145 196 394 715 Trachea 177 41863 692553 Muscle 0.52 0.64 0.74 1.1 Liver 0.47 1.9 0.83 1.2 Kidney 1.0 0.792.92 1.8 Lung 0.66 1.7 2.4 27

TABLE 4 Experimental Means of Betaseron Concentration (nM) FollowingI.N. Administration of ¹²⁵I-IFNβ + rhIFNβ Avg for 1F34,37,38 46.67 6.13microCi/nmol Mean Std Dev Blood Sample #1 1.1 0.53 Blood Sample #2 2.71.2 Blood Sample #3 4.4 1.9 Blood Sample #4 5.7 2.4 Blood Sample #5 7.13.1 Blood Sample #6 9.5 1.4 Left Olfactory Epithelium 365 290 RightOlfactory Epithelium 1128 948 Left Olfactory Bulb 3.6 2.0 RightOlfactory Bulb 5.5 3.7 Anterior Olfactory Nucleus 1.6 0.81 FrontalCortex 0.70 0.37 Caudate/Putamen 0.80 0.85 L+R Hippocampus 0.61 0.22Left Hippocampus Right Hippocampus Diencephalon 0.78 0.16 Midbrain 0.660.20 Pons 0.71 0.24 Medulla 0.69 0.30 Cerebellum 0.52 0.17 Ventral BrainDura 11 4.3 Optic Nerve + Chiasm 10 13 Trigeminal Nerve 11 7.5 SpinalDura 0.61 0.49 Upper Cervical Cord 0.83 0.36 Cervical Spinal Cord 0.650.59 Thoracic Spinal Cord 0.35 0.23 Lumbar Spinal Cord 0.35 0.17Superficial Nodes 6.0 2.1 Left Superficial Cervical Node RightSuperficial Cervical Node Deep Cervical Nodes 39 41 Left Deep CervicalNode Right Deep Cervical Node Common Carotids 25 12 Thyroid 602 309Esophagus 418 286 Trachea 474 266 Muscle 0.78 0.27 Liver 0.82 0.35Kidney 1.9 0.95 Lung 10 15

TABLE Summary of Betaseron Concentration (nM) in the Cervical LymphNodes Following I.N. Administration of ¹²⁵I-IFN-β + rhIFN-β ExperimentIF34 IF36 IF37 IF38 MicroCi 31 47 61 Nmol 3.9 6.9 7.9 6.6 Average StdDev Superficial 8.1 6.3 4.0 6.1 6.1 1.7 Cervical Nodes Deep Cervical 9.716 68 31 32 NodesAverage Dose Administered = 46.75 uCi and 6.32 nmol

Example 4 Intravenous Administration of Betaseron

Intravenous administration of Betaseron was studied in order todetermine the extent to which delivery to the CNS and/or lymphaticsystem following intranasal administration may be due to absorption fromthe nasal cavity into the circulation, followed by subsequent deliveryto the CNS and lymphatics.

Male Harlan Sprague-Dawley rats weighing 263-318 g were used for theseexperiments. Rats were anaesthetized with sodium pentobarbital(Nembutal, 50 mg/kg). For each rat, a 500 μl solution containing¹²⁵I-IFN-β and rhIFN-β in 0.9% NaCl was delivered intravenously over60-90 seconds through a cannula into the femoral vein. On average, 560pmol and 49 μCi of IFN-β were administered to each rat. Then 0.2 ml ofblood was collected from the descending aorta cannula every 5 minutesfor a total of five blood samples. Lastly, the rat was perfused throughthe descending aorta cannula with 60-90 ml of 0.9% NaCl followed by 400ml of fixative (4% paraformaldehyde in Sorenson's phosphate buffer).Individual tissue sections were dissected out, placed in 5 ml Startedttubes, and then counted for gamma rays in the Packard Cobra II autogammacounter.

The methods described above created the same general blood level ofBetaseron with intravenous delivery as that achieved in the intranasaladministration studies. Tables 6 and 7 provide the level of Betaseron inthe blood following either intravenous injection and intranasaladministration. The level of Betaseron in the blood followingintravenous administration and intranasal administration over time isshown graphically in FIG. 1.

This study demonstrated that very little of the intravenouslyadministered Betaseron reaches either the CNS or lymphatics.Consequently, it is clear that the intranasal method of deliverydescribed in this application is very beneficial in targeting the CNSand lymphatics of the head and neck region. This method of delivery doesnot utilize the circulation to reach the CNS or lymphatics, but ratherbypasses the circulation and blood-brain barrier to accomplish delivery.Because it is not necessary to use the circulatory system to deliver themedication to the CNS and/or lymphatics, systemic side effects can besignificantly reduced. TABLE 6 Level of Betaseron in the blood followingintravenous administration. Experiment # IF47 IF49 IF50 Mean Std ErrDelivered nmol 0.521 0.579 0.579 0.560 0.019 Delivered uCi 56 46 45 49 45 min Blood Sample 6.30 4.47 6.78 5.85 0.70 10 min Blood Sample 5.354.41 5.29 5.01 0.30 15 min Blood Sample 5.90 4.14 5.95 5.33 0.60 20 minBlood Sample 5.95 4.48 5.92 5.45 0.49 25 min Blood Sample 6.40 4.16 6.305.62 0.73

TABLE 7 Level of Betaseron in the blood stream following intravenousadministration. Experiment # IF36 IF37 IF38 IF40 Mean Std Err Deliverednmol 6.890 7.947 6.583 7.360 7.195 0.30 Delivered uCi 47 61 48 51 52 3 5min Blood 0.58 1.08 1.60 2.44 1.43 0.40 Sample 10 min Blood 1.43 2.763.89 5.91 3.50 0.95 Sample 15 min Blood 3.06 4.62 6.22 8.30 5.55 1.12Sample 20 min Blood 4.70 5.14 8.27 10.17 7.07 1.30 Sample 25 min Blood6.93 7.04 10.16 12.85 9.25 1.42 Sample

TABLE 8 Concentration (nM) following intravenous administration ofIFN-β. IF47 IF49 IF50 Mean Std Err Delivered nmol 0.521 0.579 0.5790.560 0.019 Delivered uCi 56 46 45 49 4 Blood Sample#1 (5 min) 6.30 4.476.78 5.85 0.70 Blood Sample#2 5.35 4.41 5.29 5.02 0.30 (10 min) BloodSample#3 5.90 4.14 5.95 5.33 0.60 (15 min) Blood Sample#4 5.95 4.47 5.925.45 0.49 (20 min) Blood Sample#5 6.40 4.16 6.30 5.62 0.73 (25 min) LeftOlfactory 0.27 0.72 0.86 0.62 0.18 Epithelium Right Olfactory 0.19 0.781.04 0.67 0.25 Epithelium Left Olfactory Bulb 0.63 0.23 0.31 0.39 0.12Right Olfactory Bulb 1.01 0.22 0.23 0.49 0.26 Anterior Olfactory 0.150.13 0.17 0.15 0.01 Nucleus Frontal Cortex 0.15 0.16 0.18 0.16 0.01Caudate/Putamen 0.21 0.11 0.15 0.16 0.03 Hippocampus 0.13 0.11 0.14 0.130.01 Cerebellum 0.15 0.12 0.16 0.14 0.01 Diencephalon 0.14 0.12 0.140.13 0.01 Midbrain 0.16 0.12 0.14 0.14 0.01 Pons 0.13 0.11 0.03 0.090.03 Medulla 0.14 0.11 0.14 0.13 0.01 Dorsal Brain Dura 0.41 0.43 0.420.01 Ventral Brain Dura 1.32 0.28 0.17 0.59 0.37 Optic Nerve + Chiasm0.18 0.29 0.24 0.04 Trigeminal Nerve 0.28 0.21 0.26 0.25 0.02 SpinalDura 0.07 0.13 0.12 0.11 0.02 Upper Cervical Cord 0.15 0.12 0.09 0.120.02 Cervical Cord 0.10 0.10 0.09 0.10 0.00 Thoracic Spinal Cord 0.080.09 0.11 0.09 0.01 Lumbar Spinal Cord 0.11 0.12 0.14 0.12 0.01Superficial Nodes 0.42 0.28 0.64 0.45 0.10 Deep Cervical Nodes 0.10 0.340.40 0.28 0.09 Axial Nodes 0.33 0.64 0.49 0.13 Common Carotids 0.13 0.110.09 0.11 0.01 Thyroid 52.65 56.49 11.03 40.06 14.56 Esophagus 0.92 0.910.29 0.71 0.21 Trachea 0.81 0.49 0.46 0.59 0.11 Deltoid Muscle 0.29 0.190.30 0.26 0.04 Liver 15.17 11.82 16.90 14.63 1.49 Kidney 1.30 1.51 1.491.43 0.07 Lung 16.02 30.02 33.14 26.39 5.26

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1-20. (canceled)
 21. A method for administering human IFN-β or a variantthereof to a central nervous system or a lymphatic system of a mammal,comprising: administering a pharmaceutical composition comprising aneffective amount of the IFN-β or a variant thereof to a tissueinnervated by the trigeminal nerve and outside of the nasal cavity,wherein the IFN-β3 or a variant thereof is absorbed through the tissueof the mammal in an amount effective to reduce or treat an infection ordisorder is selected from viral meningitis herpes simplex, hepatitis-C,HIV, multiple sclerosis, and a glioma, and wherein said variant has atleast 70% sequence identity to the human interferon-β and retainsantiviral activity or anti-proliferative activity.
 22. The method ofclaim 21, wherein the tissue comprises a skin.
 23. The method of claim22, wherein administering the cytokine to the skin comprisesadministering the cytokine to a face, a forehead, an upper eyelid, alower eyelid, a dorsum of the nose, a side of the nose, an upper lip, acheek, a chin, a scalp, or a combination thereof.
 24. The method ofclaim 21, wherein the tissue comprising a conjunctiva.
 25. The method ofclaim 24, wherein the tissue comprises the conjunctive is the mucosa ofthe upper or lower eyelid.
 26. The method of claim 21, wherein thetissue comprises an oral tissue.
 27. The method of claim 26, wherein theoral tissue is a gingiva tissue.
 28. The method of claim 26, wherein theoral tissue is the anterior two-thirds of the tongue.
 29. The method ofclaim 26, wherein the oral tissue is the mucosa of a cheek.
 30. Themethod of claim 26, wherein the oral tissue is the mucosa of a upper orlower lip.
 31. The method of claim 26, wherein administering thecytokine to the oral tissue comprises sublingual administration. 32.(canceled)
 33. The method of claim 21, wherein said IFN-β is the maturehuman IFN-β or a biologically active variant thereof.
 34. (canceled) 35.The method of claim 21, wherein the IFN-β or variant thereof istransported to the central nervous system of the mammal.
 36. The methodof claim 35, wherein said infection or disorder is selected from thegroup consisting of viral meningitis, herpes simplex, hepatitis C, andhuman immunodeficiency (HIV).
 37. (canceled)
 38. The method of claim 35,wherein said infection or disorder is selected from the group consistingof meningitis, multiple sclerosis, and HIV.
 39. (canceled)
 40. Themethod of claim 35, wherein said infection or disorder is a glioma. 41.The method of claim 21, wherein the IFN-β or variant thereof isadministered in a dosage range from about 0.14 nmol/kg of brain weightto about 138 nmol/kg of brain weight.
 42. (canceled)
 43. The method ofclaim 21, wherein the IFN-β or variant thereof is transported to thelymphatic system of the mammal in an amount effect.
 44. The method ofclaim 43, wherein said infection or disorder is selected from the groupconsisting of viral meningitis, herpes simplex, hepatitis C, and humanimmunodeficiency (HIV).
 45. (canceled)
 46. The method of claim 43,wherein said infection or disorder is selected from the group consistingof meningitis, multiple sclerosis, and HIV.
 47. (canceled)
 48. Themethod of claim 45, wherein said infection or disorder is a glioma.